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open access article This paper was produced by the International Brain Initiative Data Standards and Sharing Working Group's Taskforce on International Data Governance chaired by Damian Eke (from CCSR) As neuroscience projects increase in scale and cross international borders, different ethical principles, national and international laws, regulations, and policies for data sharing must be considered. These concerns are part of what is collectively called data governance. Whereas neuroscience data transcend borders, data governance is typically constrained within geopolitical boundaries. An international data governance framework and accompanying infrastructure can assist investigators, institutions, data repositories, and funders with navigating disparate policies. Here, we propose principles and operational considerations for how data governance in neuroscience can be navigated at an international scale and highlight gaps, challenges, and opportunities in a global brain data ecosystem. We consider how to approach data governance in a way that balances data protection requirements and the need for open science, so as to promote international collaboration through federated constructs such as the International Brain Initiative (IBI).

The International Brain Initiative (IBI) has been established to coordinate efforts across existing and emerging national and regional brain initiatives. This NeuroView describes how to be involved and the new opportunities for global collaboration that are emerging between scientists, scientific societies, funders, industry, government, and society.

Introduction Numerous studies have shown that nicotine (NIC) can enhance the reinforcing effects of non-NIC stimuli through a nonassociative mechanism. To date, it is unclear whether NIC reinforcement enhancement serves to increase behaviors motivated by rewarding stimuli only, or whether NIC potentiates behavior motivated by all stimuli, regardless of valence. Methods The current study used a place conditioning procedure to examine whether acute NIC injection modulates avoidance of an environment previously associated with an aversive stimulus. Separate groups of rats underwent place conditioning using either lithium chloride (125mg/kg/ml, i.p.) or footshock (0.75 mA) as the aversive stimulus. Other rats served as nonconditioned controls. The magnitude of place avoidance was assessed after acute NIC (0.1 or 0.4mg/kg/ml, s.c.) or saline. Results Rats avoided chambers previously paired with either lithium chloride or footshock, and conditioned place avoidance was significantly enhanced by NIC pre-treatment. Conclusions These results demonstrate that the ability of NIC to enhance motivated behavior extends to behaviors elicited by aversive stimuli, evidence that NIC affects behavior motivated by a broader range of stimuli than previously appreciated. Implications The current study examined whether the reinforcement enhancement properties of NIC apply to aversive stimuli by testing NIC enhancement of conditioned place avoidance in rats. The results demonstrate that NIC enhances the motivational impact of these distinct aversive stimuli, providing novel evidence that NIC affects behavior motivated by a broader range of stimuli than has previously been demonstrated.

Abnormal levels of dopamine (DA) are thought to contribute to several neurological and psychiatric disorders including drug addiction. Extracellular DA levels are regulated primarily via reuptake by the DA transporter (DAT). Amphetamine, a potent psychostimulant, increases extracellular DA by inducing efflux through DAT. Recently, we discovered that G protein βγ subunits (Gβγ) interact with DAT, and that in vitro activation of Gβγ promotes DAT-mediated efflux. Here, we investigated the role of Gβγ in the actions of amphetamine in DA neurons in culture, ex vivo nucleus accumbens (NAc), and freely moving rats. Activation of Gβγ with the peptide myr-Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp (mSIRK) in the NAc potentiated amphetamine-induced hyperlocomotion, but not cocaine-induced hyperlocomotion, and systemic or intra-accumbal administration of the Gβγ inhibitor gallein attenuated amphetamine-induced, but not cocaine-induced hyperlocomotion. Infusion into the NAc of a TAT-fused peptide that targets the Gβγ-binding site on DAT (TAT-DATct1) also attenuated amphetamine-induced but not cocaine-induced hyperlocomotion. In DA neurons in culture, inhibition of Gβγ with gallein or blockade of the Gβγ–DAT interaction with the TAT-DATct1 peptide decreased amphetamine-induced DA efflux. Furthermore, activation of Gβγ with mSIRK potentiated and inhibition of Gβγ with gallein reduced amphetamine-induced increases of extracellular DA in the NAc in vitro and in freely moving rats. Finally, systemic or intra-accumbal inhibition of Gβγ with gallein blocked the development of amphetamine-induced, but not cocaine-induced place preference. Collectively, these results suggest that interaction between Gβγ and DAT plays a critical role in the actions of amphetamine and presents a novel target for modulating the actions of amphetamine in vivo.

Previous work demonstrates that spatial (explicit) and nonspatial (implicit) elements of place learning in the Morris water maze (MWM) task can be dissociated and examined in the context of experimental traumatic brain injury (TBI). Providing nonspatial cognitive training (CT) after injury can improve place learning compared with untrained controls. In the present study, we hypothesized that brief exposure to extra-maze cues, in conjunction with CT, may further improve MWM performance and extra-maze cue utilization compared with CT alone. Adult male Sprague-Dawley rats (n = 66) received controlled cortical impact (CCI) injury or sham surgery. Beginning day 8 postsurgery, CCI and sham rats received 6 days of no training (NT) or CT with/without brief, noncontextualized exposure to extra-maze cues (BE and CT, respectively). Acquisition (days 14-18), visible platform (VP; day 19), carryover (CO; days 20-26), and periodic probe trials were performed. Platform latencies, peripheral and target zone time allocation, and search strategies were assessed. CCI/BE rats had shorter acquisition trial latencies than CCI/NT ( P < .001) and tended to have shorter latencies than CCI/CT rats ( P < .10). Both BE and CT reduced peripheral zone swimming for CCI rats versus CCI/NT. CCI/BE animals increased spatial swim strategies from day 14 to day 18 relative to CCI/CT and showed similar swim strategy selection to the Sham/NT group. These data suggest that visual priming improves initial place learning in the MWM. These results support the visual priming response as another clinically relevant experimental rehabilitation construct, to use when assessing injury and treatment effects of behavioral and pharmacological therapies on cognition after TBI.

Background. Following traumatic brain injury (TBI), clinical cognitive training paradigms harness implicit and explicit learning and memory systems to improve function; however, these systems are differentially affected by TBI, highlighting the need for an experimental TBI model that can test efficacy of cognitive training approaches. Objectives. To develop a clinically relevant experimental cognitive training model using the Morris water maze (MWM) wherein training on implicitly learned task components was provided to improve behavioral performance post-TBI. Methods. Eighty-one adult male rats were divided by injury status (controlled cortical impact [CCI]/Sham), non-spatial cognitive training (CogTrained/No-CogTrained), and extra-maze cues (Cued/Non-Cued) during MWM testing. Platform latencies, thigmotaxis, and search strategies were assessed during MWM trials. Results. Cognitive training was associated with improved platform latencies, reduced thigmotaxis, and more effective search strategy use for Sham and CCI rats. In the Cued and Non-Cued MWM paradigm, there were no differences between CCI/CogTrained and Sham/No-CogTrained groups. During novel testing conditions, CogTrained groups applied implicitly learned knowledge/skills; however, sham-cued CogTrained/rats better incorporated extramaze cues into their search strategy than the CCI-Cued group. Cognitive training had no effects on contusion size or hippocampal cell survival. Conclusions. The results provide evidence that CCI-CogTrained rats that learned the nonspatial components of the MWM task applied these skills during multiple conditions of the place-learning task, thereby mitigating cognitive deficits typically associated with this injury model. The results show that a systematic application of clinically relevant constructs associated with cognitive training paradigms can be used with experimental TBI to affect place learning.

Nicotine has been shown to enhance the motivational properties of non-nicotine stimuli. This reinforcement-enhancing property of nicotine has the potential to promote the use of other illicit substances as well as maladaptive patterns of food intake. Therefore, the current study aimed to examine whether nicotine enhances preference for contexts paired with cocaine or sucrose utilizing a place conditioning procedure. Separate groups of adult male rats were administered sucrose or cocaine in one of two compartments of a standard CPP chamber on four consecutive days. Preference was then assessed following no injection, a single subcutaneous (s.c.) injection of nicotine, and a s.c. saline injection. The animals preferred the chamber paired with either sucrose or cocaine, as evident from an increased time spent in the paired chamber compared to baseline. Nicotine further increased the time spent in the sucrose- or cocaine-paired chamber, consistent with a reinforcement-enhancement effect. Previous results demonstrate an interaction between nicotine and intake of other drugs or food. The present findings provide an additional mechanism that may underlie these effects and which may have implications for drug dependence and obesity.

Explicit and implicit learning and memory networks exist where each network can facilitate or inhibit cognition. Clinical evidence suggests that implicit networks are relatively preserved after traumatic brain injury (TBI). Non-spatial pre-training (NSPT) in the Morris Water Maze (MWM) provides the necessary behavioral components to complete the task, while limiting the formation of spatial maps. Our study utilized NSPT in the MWM to assess implicit and explicit learning and memory system deficits in the controlled cortical impact (CCI) model of TBI. 76 adult male Sprague–Dawley rats were divided: CCI vs. sham surgery, NSPT vs. No-NSPT, and cued vs. non-cued groups. NSPT occurred for 4d prior to surgery (dynamic hidden platform location, extra-maze cues covered, static pool entry point). Acquisition (d14–18), Probe/Visible Platform (d19), and Reversal (d20–21) trials were conducted with or without extra-maze cues. Novel time allocation and search strategy selection metrics were utilized. Results indicated implicit and explicit learning/memory networks are distinguishable in the MWM. In the cued condition, NSPT reduced thigmotaxis, improved place learning, and largely eliminated the apparent injury-induced deficits typically observed between untrained CCI and sham rats. However, among NSPT groups, incorporation of cues into search strategy selection for CCI rats was relatively impaired compared to shams. Non-cued condition performance showed sham/NSPT and CCI/NSPT rats perform similarly, suggesting implicit memory networks are largely intact 2 weeks after CCI. Place learning differences between CCI/NSPT and sham/NSPT rats more accurately reflect spatial deficits in our CCI model compared to untrained controls. These data suggest NSPT as a clinically relevant construct for evaluating potential neurorestorative and neuroprotective therapies. These findings also support development of non-spatial cognitive training paradigms for evaluating rehabilitation relevant combination therapies.

The field of neuroscience is enjoying a rapid expansion in scope, coupled with a remarkable broadening of conceptual approaches, scientific tools, and clinical applications. This growth poses new challenges for academic training programs as they prepare young neuroscientists for a more complex, competitive, and diverse career landscape. Multiple stakeholders, including academia, federal funding agencies, industry, scientific societies, private foundations, and other public and private sector contributors, need to be actively engaged in supporting this broad training effort. A renewed commitment to a more forward-looking, flexible yet integrative training vision offers opportunities for a bright future for young neuroscientists as they assume the role of vanguard of the enterprise that enriches our understanding of the brain.

The transcription factor cAMP response element-binding protein (CREB) in the nucleus accumbens (NAc) has been shown to regulate an animal’s behavioral responsiveness to emotionally salient stimuli, and an increase in CREB phosphorylation in the NAc has been observed during exposure to rewarding stimuli, such as drugs of abuse. Here we show that CREB phosphorylation increases in the NAc also during exposure to cues that an animal has associated with delivery of natural rewards. Adult male Sprague–Dawley rats (rattus norvegicus) were trained to associate an auditory stimulus with delivery of food pellets, and CREB phosphorylation was examined in the striatum following training. We found that repeated tone-food pairings resulted in an increase in CREB phosphorylation in the NAc but not in the adjacent dorsal striatum or in the NAc 3 h after the final training session. We further found that the cue itself, as opposed to the food pellets, the training context, or tone-food pairings, was sufficient to increase CREB phosphorylation in the NAc. These results suggest that the processing of primary rewarding stimuli and of environmental cues that predict them triggers similar accumbal signaling mechanisms.

Superoxide has been shown to be critically involved in several pathological manifestations of aging animals. In contrast, superoxide also can act as a signaling molecule to modulate signal transduction cascades required for hippocampal synaptic plasticity. Mitochondrial superoxide dismutase (SOD-2 or Mn-SOD) is a key antioxidant enzyme that scavenges superoxide. Thus, SOD-2 may not only prevent aging-related oxidative stress, but may also regulate redox signaling in young animals. We used transgenic mice overexpressing SOD-2 to study the role of mitochondrial superoxide in aging, synaptic plasticity, and memory-associated behavior. We found that overexpression of SOD-2 had no obvious effect on synaptic plasticity and memory formation in young mice, and could not rescue the age-related impairments in either synaptic plasticity or memory in old mice. However, SOD-2 overexpression did decrease mitochondrial superoxide in hippocampal neurons, and extended the lifespan of the mice. These findings increase our knowledge of the role of mitochondrial superoxide in physiological and pathological processes in the brain.

Acute amphetamine (AMPH) exposure elevates extracellular dopamine through a variety of mechanisms that include inhibition of dopamine reuptake, depletion of vesicular stores, and facilitation of dopamine efflux across the plasma membrane. Recent work has shown that the DAT substrate AMPH, unlike cocaine and other nontransported blockers, can also stimulate endocytosis of the plasma membrane dopamine transporter (DAT). Here, we show that when AMPH enters the cytoplasm it rapidly stimulates DAT internalization through a dynamin-dependent, clathrin-independent process. This effect, which can be observed in transfected cells, cultured dopamine neurons, and midbrain slices, is mediated by activation of the small GTPase RhoA. Inhibition of RhoA activity with C3 exotoxin or a dominant-negative RhoA blocks AMPH-induced DAT internalization. These actions depend on AMPH entry into the cell and are blocked by the DAT inhibitor cocaine. AMPH also stimulates cAMP accumulation and PKA-dependent inactivation of RhoA, thus providing a mechanism whereby PKA- and RhoA-dependent signaling pathways can interact to regulate the timing and robustness of AMPH’s effects on DAT internalization. Consistent with this model, the activation of D1/D5 receptors that couple to PKA in dopamine neurons antagonizes RhoA activation, DAT internalization, and hyperlocomotion observed in mice after AMPH treatment. These observations support the existence of an unanticipated intracellular target that mediates the effects of AMPH on RhoA and cAMP signaling and suggest new pathways to target to disrupt AMPH action.

INTRODUCTION Although nearly 90% of current smokers initiated tobacco use during adolescence, little is known about reinforcement by nicotine in adolescents. Researchers are currently investigating whether a potential public health policy setting a tobacco product standard with very low nicotine levels would improve public health, and it is essential to understand whether data generated in adults translates to adolescents, particularly as it relates to the threshold dose of nicotine required to support smoking. The present study compared self-administration of low doses of nicotine between adolescent and adult rats. METHODS Adolescent (postnatal day [P] 30) and adult (P90) male and female rats were allowed to nosepoke to receive intravenous infusions of nicotine (3-100 μg/kg/infusion) during 16 daily 1-hour sessions. RESULTS At 10 μg/kg/infusion nicotine, adolescent rats earned significantly fewer infusions than adults. When responding for 30 μg/kg/infusion nicotine, rats of both ages earned a similar number of infusions; however, there were subtle differences in the distribution of infusions across the 1-hour session. No sex differences were apparent in either age group at any dose. CONCLUSIONS These results demonstrate that adolescent rats are less sensitive than adults to the primary reinforcing effects of nicotine. However, at nicotine doses that support self-administration in both age groups, adolescent and adult rats do not differ in acquisition or number of infusions earned. These results suggest that reducing nicotine levels in cigarettes to a level that does not support smoking in adults may be sufficient to reduce the acquisition of smoking in adolescents. IMPLICATIONS The results of the present studies demonstrate that adolescent rats are less sensitive than adults to the primary reinforcing effects of nicotine. These results suggest that reducing nicotine levels in cigarettes to a level that does not support smoking in adults will be sufficient to reduce the acquisition of smoking in adolescents.

Cortical excitatory and inhibitory synapses are disrupted in schizophrenia, the symptoms of which often emerge during adolescence, when cortical excitatory synapses undergo pruning. In auditory cortex, a brain region implicated in schizophrenia, little is known about the development of excitatory and inhibitory synapses between early adolescence and young adulthood, and how these changes impact auditory cortex function. We used immunohistochemistry and quantitative fluorescence microscopy to quantify dendritic spines and GAD65-expressing inhibitory boutons in auditory cortex of early adolescent, late adolescent, and young adult mice. Numbers of spines decreased between early adolescence and young adulthood, during which time responses increased in an auditory cortex-dependent sensory task, silent gap-prepulse inhibition of the acoustic startle reflex (gap-PPI). Within-bouton GAD65 protein and GAD65-expressing bouton numbers decreased between late adolescence and young adulthood, a delay in onset relative to spine and gap-PPI changes. In mice lacking the spine protein kalirin, there were no significant changes in spine number, within-bouton GAD65 protein, or gap-PPI between adolescence and young adulthood. These results illustrate developmental changes in auditory cortex spines, inhibitory boutons, and auditory cortex function between adolescence and young adulthood, and provide insights into how disrupted adolescent neurodevelopment could contribute to auditory cortex synapse pathology and auditory impairments.

The neural substrates of memory likely include long-term potentiation (LTP) of synaptic strength that results from high-frequency stimulation (HFS) of the afferent pathway. The mechanisms that underlie the maintenance of LTP include RNA and protein synthesis, although the contribution of these molecular events typically does not become essential until several hours after LTP induction. We here show that, different from this pattern, (1) LTP maintenance at the mossy fiber (MF) input to CA3 pyramidal cells in the hippocampus depends on protein and RNA synthesis soon after LTP induction, and (2) some of these molecular events are controlled by signaling from the presynaptic granule cell soma. Bath application of the protein synthesis inhibitor emetine or cycloheximide 1 hr after MF LTP induction in hippocampal slices caused loss of MF potentiation. In contrast, application of emetine 1 hr after LTP induction at the commissural—associational input to CA3 pyramidal cells had no effect on this form of LTP. Administration of emetine or the RNA synthesis inhibitor actinomycin-D before delivery of HFS to MF input also caused a rapid decay of MF potentiation, although neither drug had an effect on the amplitude or the time-constant of decay of post-tetanic potentiation (PTP). Similarly, transection of MF axons near granule cell somas had no effect on baseline or PTP parameters but caused loss of potentiation at a rate comparable with that after actinomycin-D application. These results indicate that the mechanisms that underlie MF LTP maintenance differ from those involved in LTP maintenance at other glutamatergic synapses.

The reactive oxygen species (ROS) superoxide is well known for its role in disease mechanisms. Mounting evidence indicates, however, that superoxide also is generated for useful purposes and contributes to normal physiologic function. Studies with transgenic mice that overexpress superoxide scavengers show that certain types of memory function and underlying neuronal processes are impaired under conditions of severely reduced superoxide signaling. These findings have implications for the use of antioxidant treatments as well as for our understanding of the signaling events involved in cognition.

Protein kinase cascades likely play a critical role in the signaling events that underlie synaptic plasticity and memory. The extracellular signal-regulated kinase (ERK) cascade is suited well for such a role because its targets include regulators of gene expression. Here we report that the ERK cascade is recruited during long-term depression (LTD) of synaptic strength in area CA1 of the adult hippocampusin vivoand selectively impacts on phosphorylation of the nuclear transcription factor Elk-1. Using a combination ofin vivoelectrophysiology, biochemistry, pharmacology, and immunohistochemistry, we found the following: (1) ERK phosphorylation, including phosphorylation of nuclear ERK, and ERK phosphotransferase activity are increased markedly, albeit transiently, after the induction of NMDA receptor-dependent LTD at the commissural input to area CA1 pyramidal cells in the hippocampus of anesthetized adult rats; (2) LTD-inducing paired-pulse stimulation fails to produce lasting LTD in the presence of the ERK kinase inhibitor SL327, which suggests that ERK activation is necessary for the persistence of LTD; and (3) ERK activation during LTD results in increased phosphorylation of Elk-1 but not of the transcription factor cAMP response element-binding protein. Our findings indicate that the ERK cascade transduces signals from the synapse to the nucleus during LTD in hippocampal area CA1in vivo, as it does during long-term potentiation in area CA1, but that the pattern of coupling of the ERK cascade to transcriptional regulators differs between the two forms of synaptic plasticity.

Conditioned stimuli (CS) can modulate reward-seeking behavior. This modulatory effect can be maladaptive and has been implicated in excessive reward seeking and relapse to drug addiction. We previously demonstrated that exposure to an appetitive CS causes an increase in the activation of extracellular signal-regulated kinase (ERK) and cyclic-AMP response-element binding protein (CREB) in the nucleus accumbens (NAc) of rats, and that CS-evoked ERK activation is critical for CS control over reward seeking. To elucidate the mechanism that mediates CS-driven ERK activation in the NAc, we selectively blocked NMDA glutamate or D1 dopamine receptors in the NAc. To determine whether CS-driven ERK and CREB activation are linked, we selectively blocked ERK signaling in the NAc. We found that both NMDA and D1 receptors are critical for CS-driven ERK signaling in the NAc, and that this recruitment of the ERK cascade is responsible for increased CREB activation in the presence of the CS. Our findings suggest that activation of the NMDAR-D1R/ERK/CREB signal transduction pathway plays a critical role in the control of reward-seeking behavior by reward-predictive cues.

Acute amphetamine (AMPH) exposure elevates extracellular dopamine by a variety of mechanisms including increasing the rate of internalization of the plasma membrane dopamine transporter (DAT). We report here that AMPH also activates the small GTPases, Rho and Rac. In recombinant expression systems, primary cultures and midbrain slices, activation of Rho and the downstream Rho-kinase (ROCK) triggers endocytosis of DAT. Inhibition of RhoA activity blocks AMPH-induced DAT internalization, while Rac1 inhibition has no significant effect on DAT trafficking. ROCK inhibitors also prevent AMPH-induced DAT internalization. Intriguingly, we found that AMPH must enter the cell to initiate this cascade. Further, intracellular AMPH also increases cAMP, leading to the phosphorylation of Rho by protein kinase A (PKA) that inactivates Rho and serves as a break on DAT internalization, thus demonstrating an important interaction between PKA- and RhoA-dependent signaling in mediating the actions of AMPH. In agreement with o...

There is growing evidence that activation of either protein kinases or protein phosphatases determines the type of plasticity observed after different patterns of hippocampal stimulation. Because activation of the extracellular signal-regulated kinase (ERK) has been shown to be necessary for long-term potentiation, we investigated the regulation of ERK in long-term depression (LTD) in the adult hippocampus in vivo. We found that ERK immunoreactivity was decreased following the induction of LTD and that this decrease required NMDA receptor activation. The LTD-associated decrease in ERK immunoreactivity could be simulated in vitro via incubation of either purified ERK2 or hippocampal homogenates with either protein phosphatase 1 or protein phosphatase 2A. The protein phosphatase-dependent decrease in ERK immunoreactivity was inhibited by microcystin. Intrahippocampal administration of the protein phosphatase inhibitor okadaic acid blocked the LTD-associated decrease in ERK2, but not ERK1, immunoreactivity. Collectively, these data demonstrate that protein phosphatases can decrease ERK immunoreactivity and that such a decrease occurs with ERK2 during LTD. These observations provide the first demonstration of a biochemical alteration of ERK in LTD.

The neural substrates of learning and memory are thought to involve use-dependent long-term changes in synaptic function, including long-term depression (LTD) of synaptic strength. One biochemical event hypothesized to contribute to the maintenance and expression of LTD is decreased protein phosphorylation, caused by a decrease in protein kinase activity and/or an increase in protein phosphatase activity. We tested whether the activity of protein kinase C (PKC) decreases after the induction of LTD in area CA1 of the adult hippocampus in vivo, and then investigated the mechanism responsible for the LTD-associated alteration in PKC activity. We found that LTD was associated with a significant decrease in both autonomous and cofactor-dependent PKC activity. The decrease in PKC activity was prevented by NMDA receptor blockade and was not accompanied by a decrease in the level of either PKCalpha, beta, gamma, or zeta. Western blot analysis with phosphospecific antibodies revealed that phosphorylation of Ser-657 on the catalytic domain of PKCalpha (Ser-660 on PKCbetaII) was decreased significantly after the induction of LTD, and that this dephosphorylation was prevented by the protein phosphatase inhibitor okadaic acid. The decrease in autonomous and cofactor-dependent PKC activity likewise was prevented by okadaic acid. These findings suggest that LTD in the adult hippocampus in vivo involves a decrease in PKC activity that is mediated, at least in part, by dephosphorylation of the catalytic domain of PKC by protein phosphatases activated after LTD-inducing stimulation. Our findings are consistent with the idea that protein dephosphorylation contributes to the expression of LTD.

1. Reactive oxygen species are known for their role in neurotoxicity. However, recent studies indicate that reactive oxygen species also play a role in cell function under physiological conditions. 2. Both superoxide and hydrogen peroxide alter the activity of various protein kinases and protein phosphatases, some of which are involved in hippocampal synaptic plasticity. Specifically, the activity of protein kinase C, extracellular-regulated kinase 2, and a protein tyrosine kinase(s) is increased in the presence of these reactive oxygen species, whereas the activity of protein phosphatases 2A and 2B, and a protein tyrosine phosphatase(s) is decreased. 3. Protein kinase C, extracellular-regulated kinase 2, and protein tyrosine kinases critically participate in the induction and/or early expression of long-term potentiation at glutamatergic synapses in hippocampus. Protein phosphatases 2A and 2B participate in the induction and/or early expression of long-term depression at these synapses. 4. Treatment of hippocampal slices with scavengers of either superoxide or hydrogen peroxide prevents the full expression of long-term potentiation. Long-term potentiation in hippocampus also is attenuated in transgenic mice that overexpress Cu/Zn superoxide dismutase. 5. The link between reactive oxygen species and long-term potentiation may be the activating effect on protein kinases. The inhibiting effect of reactive oxygen species on protein phosphatases may also contribute to long-term potentiation. 6. The authors hypothesize that reactive oxygen species play a critical role in hippocampal long-term potentiation by favoring the activation of a protein kinase over a protein phosphatase signaling cascade.

Memantine and ketamine block N-methyl-D-aspartate (NMDA) receptors with similar affinity and kinetics, yet their behavioral consequences differ: e.g., memantine is used to alleviate symptoms of Alzheimer's disease, whereas ketamine reproduces symptoms of schizophrenia. The two drugs exhibit different pharmacokinetics, which may play a principal role in their differential behavioral effects. To gain insight into the drugs' behavioral consequences, we treated adult male rats acutely with varying doses (0-40 mg/kg i.p.) of memantine or ketamine and assessed exploratory behavior and spatial working memory. To examine the importance of pharmacokinetics, we assessed behavior either 15 or 45 min after drug administration. Both drugs decreased ambulation, fine movements, and rearing at the beginning of the exploratory activity test; however, at the end of the test, high doses of only memantine increased ambulation and fine movements. High doses of both drugs disrupted spontaneous alternation, a measure of working memory, but high doses of only memantine elicited perseverative behavior. Surprisingly, ketamine's effects were influenced by the delay between drug administration and testing no more frequently than were memantine's. Our findings show that, regardless of test delay, memantine and ketamine evoke similar behavioral effects at lower doses, consistent with NMDA receptors being both drugs' principal site of action, but can have divergent effects at higher doses. Our results suggest that the divergence of memantine's and ketamine's behavioral consequences is likely to result from differences in mechanisms of NMDA receptor antagonism or actions at other targets.

The ability of reward-predictive cues to potentiate reward-seeking behavior--a phenomenon termed Pavlovian--instrumental transfer (PIT)--depends on the activation of extracellular signal-regulated kinase (ERK) in the nucleus accumbens (NAc). Here, we utilized immunohistochemistry to investigate the subregional pattern of ERK activation during PIT, and the contribution of different elements in the PIT condition to the distribution of ERK signaling in the NAc of rats. We found that the occurrence of reward-seeking behavior (lever pressing) did not affect ERK activation in either the core or the shell of the NAc. In contrast, presentation of the reward-predictive cue (auditory conditioned stimulus) caused a significant increase in ERK activation in both subregions of the NAc, with the effect being slightly more robust in the core than the shell. Different from the pattern evoked by the reward-predictive cue, presentation of the reward itself (food pellets) had no effect on ERK activation in the core but caused a pronounced increase in ERK activation in the shell. Taken together, our results demonstrate that ERK signaling in the NAc during PIT involves both the core and the shell and is driven by the conditioned cue irrespective of whether the situation permits engagement in reward-seeking behavior. Furthermore, our results show that the subregional distribution of ERK signaling in the NAc evoked by rewards differs from that evoked by cues that predict them. The stimulus-specific differential pattern of ERK signaling described here may present the molecular complement to stimulus-specific increases in NAc cell firing reported previously.

1. As part of an effort to evaluate the biological plausibility of theoretically derived principles of synaptic modification, we studied activity-dependent long-term depression (LTD) of glutamatergic transmission in the hippocampus of anesthetized adult rats. Field potentials of CA1 pyramidal cells evoked by single-pulse stimulation (0.1 Hz) of the commissural afferents were recorded before and after paired-pulse stimulation (0.5 Hz) of the same pathway. A train of 150 or 200 paired pulses produced robust LTD of the commissural input to the CA1 pyramidal neurons when the interstimulus interval (ISI) of the pairs was short (25 ms) but not when the ISI was long (1,000 ms). 2. Paired-pulse stimulation with the short but not with the long ISI also was associated with pronounced inhibition of pyramidal cell firing upon the second pulse of a pair, despite the fact that the excitatory input was facilitated with the short-ISI paradigm. The inhibition of pyramidal cell activity was mediated by input to the pyramidal cells from local gamma-aminobutyric acid (GABA)-releasing interneurons activated by commissural fibers and/or CA1 recurrent collaterals, because the inhibition was eliminated by local administration of the selective GABAA receptor antagonist, bicuculline (50 microM), near the recording site. 3. Postsynaptic input from GABAergic interneurons was necessary for the induction of LTD, because short-ISI paired-pulse stimulation failed to produce LTD in the presence of bicuculline. 4. N-methyl-D-aspartate (NMDA) receptor-mediated excitation also was necessary for the induction of LTD, because administration of the selective NMDA receptor antagonist, D-2-amino-5-phosphonvaleric acid (100 microM), near the recording site prevented the development of LTD.(ABSTRACT TRUNCATED AT 250 WORDS)

Immediate early genes (IEGs) typically are the first genetic responders to a variety of cellular activations. The IEG that encodes activity-regulated cytoskeleton-associated protein (arc/arg3.1) has attracted much interest because its mRNA is transported to and translated near activated synapses. Moreover, arc has been implicated in both long-term potentiation (LTP) and long-term depression (LTD). However, little is known about the time course of altered arc expression during LTP and LTD. Here we characterized arc mRNA levels in area CA1 of the adult rat hippocampus in vivo after LTP- and LTD-inducing stimulations that were identical, except for the temporal patterning of the stimulation pulses. We observed a persistent increase in arc mRNA level during LTP. In contrast, during LTD, arc mRNA level first was decreased and then transiently increased relative to control level. These findings demonstrate that arc mRNA is regulated differently during LTP and LTD, and they provide evidence for stimulation-induced downregulation of mRNA availability during LTD. Findings of abbreviated LTD when transcription was inhibited indicate that the prolonged maintenance of the type of N-methyl-D-aspartate receptor-dependent LTD studied here requires de novo transcription. Furthermore, lack of evidence for a LTD-associated change in the mRNA level of the IEG zif268 demonstrates that the decrease in arc mRNA during LTD is not a general genetic response. Thus, the regulation of arc expression not only differs between LTP and LTD, but also diverges from that of other IEGs implicated in activity-dependent synaptic plasticity.

Evidence shows that the serine/threonine protein phosphatase 1 (PP1) plays a critical role in synaptic plasticity and memory. Little is known about the contribution of the serine/threonine protein phosphatase 2A (PP2A) to synaptic plasticity. Both protein phosphatases can target the transcription factor cAMP response element binding protein (CREB), whose phosphorylation at Ser133 we previously found to be down-regulated during long-term depression (LTD) of glutamatergic transmission in area CA1 of the adult hippocampus in vivo. Other work from our group showed that the activity of PP2A, as well as that of PP1, is increased after LTD induction in area CA1 in vivo. We therefore investigated here whether both protein phosphatases are necessary for LTD in area CA1, and whether they both are involved in the LTD-associated modification of CREB. We found that inhibition of either PP1 or PP2A interferes with the establishment of LTD. Furthermore, inhibition of either enzyme alone abrogated the LTD-associated dephosphorylation of CREB. Interestingly, inhibition of PP1 disrupted CREB dephosphosphorylation rapidly after LTD-inducing stimulation, whereas inhibition of PP2A did not blunt the CREB modification until a later time point. Thus, both PP1 and PP2A regulate CREB during LTD in area CA1, although possibly through different signaling pathways. Our results demonstrate that PP2A, similar to PP1, plays an essential role in the molecular events that underlie LTD at glutamatergic synapses in hippocampal area CA1 in vivo. We propose that one of the mechanisms through which these protein phosphatases may contribute to the prolonged maintenance of LTD is through the regulation of CREB.

Hippocampal mossy fiber (MF) synapses on area CA3 lacunosum-moleculare (L-M) interneurons are capable of undergoing a Hebbian form of NMDA receptor (NMDAR)-independent long-term potentiation (LTP) induced by the same type of high-frequency stimulation (HFS) that induces LTP at MF synapses on pyramidal cells. LTP of MF input to L-M interneurons occurs only at synapses containing mostly calcium-impermeable (CI)-AMPA receptors (AMPARs). Here, we demonstrate that HFS-induced LTP at these MF-interneuron synapses requires postsynaptic activation of protein kinase A (PKA) and protein kinase C (PKC). Brief extracellular stimulation of PKA with forskolin (FSK) alone or in combination with 1-Methyl-3-isobutylxanthine (IBMX) induced a long-lasting synaptic enhancement at MF synapses predominantly containing CI-AMPARs. However, the FSK/IBMX-induced potentiation in cells loaded with the specific PKA inhibitor peptide PKI6-22failed to be maintained. Consistent with these data, delivery of HFS to MFs synapsing onto L-M interneurons loaded with PKI6-22induced posttetanic potentiation (PTP) but not LTP. Hippocampal sections stained for the catalytic subunit of PKA revealed abundant immunoreactivity in interneurons located in strata radiatum and L-M of area CA3. We also found that extracellular activation of PKC with phorbol 12,13-diacetate induced a pharmacological potentiation of the isolated CI-AMPAR component of the MF EPSP. However, HFS delivered to MF synapses on cells loaded with the PKC inhibitor chelerythrine exhibited PTP followed by a significant depression. Together, our data indicate that MF LTP in L-M interneurons at synapses containing primarily CI-AMPARs requires some of the same signaling cascades as does LTP of glutamatergic input to CA3 or CA1 pyramidal cells.

Recent reports indicate that in male rats dehydration, LiCl, and cholecystokinin (CCK) each stimulate pituitary oxytocin (OT) secretion and also decrease gastric emptying and motility. In contrast, the present experiments demonstrate that nipple attachment and sucking by pups, a well-known stimulus for neurohypophysial secretion of OT, did not decrease gastric motility in lactating rats. Moreover, systemic injection of naloxone, which is known to potentiate the inhibitory effects of LiCl and CCK on gastric motility in male rats, had no effect on gastric motility of lactating rats while nursing. These data indicate that pituitary OT secretion from magnocellular neurons is not invariably linked to decreased gastric motility in rats. As such, our results support previous findings that inhibition of gastric motility is not secondary to the pituitary secretion of OT but allow a possible role for parvocellular oxytocinergic neurons that project from the hypothalamic paraventricular nucleus to the brain stem in the control of gastric function.

The motivation to seek out rewards can come under the control of stimuli associated with reward delivery. The ability of cues to motivate reward-seeking behavior depends on the nucleus accumbens (NAcc). The molecular mechanisms in the NAcc that underlie the ability of a cue to motivate reward-seeking are not well understood. We examined whether extracellular signal-regulated kinase (ERK), an important intracellular signaling pathway in learning and memory, has a role in these motivational processes. We first examined p42 ERK (ERK2) activation in the NAcc after rats were trained to associate an auditory stimulus with food delivery and found that, as a consequence of training, presentation of the auditory cue itself was sufficient to increase ERK2 activation in the NAcc. To examine whether inhibition of ERK in the NAcc prevents cue-induced reward-seeking, we infused an inhibitor of ERK, U0126, into the NAcc before assessing rats' instrumental responding in the presence versus absence of the conditioned cue. We found that, whereas vehicle-infused rats showed increased instrumental responding during cue presentation, rats infused with U0126 showed a profound impairment in cue-induced instrumental responding. In contrast, intra-NAcc U0126 infusion had no effect on rats' food-reinforced instrumental responding or their ability to execute conditioned approach behavior. Our results demonstrate learning-related changes in ERK signaling in the NAcc, and that disruption of ERK activation in this structure interferes with the incentive-motivational effects of conditioned stimuli. The molecular mechanisms described here may have implications for cue-elicited drug craving after repeated exposure to drugs of abuse.

In this study, litters of rat pups and their mothers were continuously video-monitored from Day 14 to Day 35 postpartum in order to describe the behavioral changes that occur during that period. Prior to the onset of solid food intake (Day 18), pups spent most of their active time suckling. During Days 18 to 26, feeding, drinking, grooming, and play-fighting rapidly became frequent daily activities, while suckling gradually began to decline. By Day 28, pups' food and water intake relative to body weight and time allocated to the new behaviors reached asymptote. Pups nevertheless continued to suckle until Day 34. These results suggest that (1) the transition from milk to solid food is embedded in a multitude of behavioral changes; (2) these behavioral changes have distinct temporal characteristics; and (3) the developmental period between Days 14 and 34 can be divided into three phases.

Lactating rats that were given free access to sodium-deficient food, water, and 0.51 M NaCl solution showed no evidence of sodium appetite. The estimated daily loss of 1-2 mEq Na in milk was replaced by basal daily intake of 2-5 ml of saline. Sodium loss in urine was minimal, but milk sodium concentration was unchanged, and pups grew normally. Saline intake was enhanced when lactating rats that had been maintained on standard laboratory chow were injected with 30% polyethylene glycol solution to reduce plasma volume but no more so than when virgin female rats or male rats were similarly colloid-treated. Lactating rats markedly increased their intake of NaCl solution after simply depriving them of dietary sodium for 4 days, whereas male and virgin female rats did not. These findings indicate that pronounced sodium appetite does not invariably accompany lactation in rats, although it can occur whenever such animals become hypovolemic or sodium deficient.

Superoxide dismutases (SODs) are the major antioxidant enzymes that inactivate superoxide and thereby control oxidative stress as well as redox signaling. Transgenic mice overexpressing different isozymes of SOD have been used to study the effect of SOD overexpression on hippocampal synaptic plasticity and hippocampus-dependent learning and memory. Studies with transgenic and wild-type animals of different ages show that the function of SOD overexpression changes across the life span of an animal, and comparisons between animals that overexpress different SOD isozymes suggest that the functional value of overexpression as well as the mechanisms through which the respective functional values are effected vary depending on isozyme. The work discussed in this review has important implications for the use of antioxidant treatments and for our understanding of the role of superoxide in physiological and pathological processes.

Bleomycin hydrolase is a multifaceted neutral cysteine protease with a suggested role in antigen presentation, homocysteine-thiolactone metabolism, and Alzheimer’s disease pathogenesis. Deletion of the protease in mice results in increased neonatal mortality and dermatopathology. Immunohistochemical and behavioral studies of BLMH knockout mice were undertaken to further evaluate the role of the protease in the brain. No gross abnormalities in the central nervous system were observed upon preliminary histological examination of B6.129Blmhtm1Geh/J null animals. However, glial fibrillary acid protein immunohistochemistry revealed a global reactive astrogliosis in the aged null animals, indicative of undefined brain pathology. The role of BLMH in the brain was further explored by characterizing the behavioral phenotype of hybrid [129S6-Blmhtm1Geh/J X B6.129 Blmhtm1Geh/J]F1 null and littermate controls using multiple behavioral paradigms. In the water maze, deletion of BLMH resulted in poorer performance during water maze probe trials without detectable effect of the mutation on sensorimotor function. In addition, no age-dependent decline in discriminative performance on probe trials was observed in null animals. These data suggest a physiological non-redundant function for BLMH in the central nervous system.

Signaling through gap junctions (electrical synapses) is important in the development of the mammalian central nervous system. Abundant between neurons during postnatal development, gap junction coupling subsequently decreases and remains low in the adult, confined to specific subsets of neurons. Here we report that developmental uncoupling of gap junctions in the rat hypothalamus in vivo and in vitro is associated with a decrease in connexin 36 (Cx36) protein expression. Both developmental gap junction uncoupling and Cx36 downregulation are prevented by the blockade of NMDA glutamate receptors, action potentials and the calcium-cyclic AMP response element binding protein (CREB), and are accelerated by CREB overexpression. Developmental gap junction uncoupling and Cx36 downregulation are not affected by blockade of non-NMDA glutamate receptors, and do not occur in hypothalamic neurons from NMDA receptor subunit 1 (NMDAR1) knockout mice. These results demonstrate that NMDA receptor activity contributes to the developmental uncoupling of gap junctions via CREB-dependent downregulation of Cx36.

The mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), has been studied extensively in recent years for its involvement in synaptic plasticity and memory function. Activation of ERK is coupled to stimulation of cell-surface proteins via several different upstream signaling pathways, and contributes to the regulation of diverse cellular processes, ranging from cell excitability to gene expression. We herein review evidence for ERK's role in different forms of synaptic plasticity and different types of learning paradigms, drawing on examples from different systems in molluscs as well as the mammalian brain. The picture that emerges is that ERK activation in response to conditions that give rise to synaptic and behavioral modification contributes to that modification in a multitude of functionally distinct ways. The functional diversity is likely to be achieved by the operation of multiple, parallel ERK cascades that differ with respect to the subcellular compartments in which ERK exerts its effects and the temporal windows during which the effects are manifested. We conclude that our understanding of the mechanisms by which ERK contributes to synaptic plasticity and memory has much to gain by further study of the signaling events up- and downstream of ERK activation and the spatiotemporal characteristics of ERK activation in association with activity-dependent synaptic modification and information processing.

s / Drug and Alcohol Dependence 140 (2014) e169–e251 e213 drugs were dropped from analyses due to insufficient sample size (n=15). Our outcome variables were husband (H) to wife (W) and W to H IPV. We employed multivariate Poisson multilevel models, simultaneously examining bothH andW IPV.We adjusted drug use estimates for sociodemographic variables, heavy alcohol use, and antisocial behavior. Results: In a main effects-only model, H other drug use significantly predicted W to H IPV [incidence rate ratio (IRR) =2.29; p

In the first experiment, 4 pigeons were each presented with a recurring sequence of four key colors followed by the delivery of grain (block clock). Once the rate of pecking had stabilized, three of the colors were replaced, during different series of sessions, by a darkening of the key. The rate of pecking was reduced within those segments of the interval between deliveries of food during which the key was dark; when the key was dark during the final portion of the interval, rates were reduced throughout the entire interval. In the second experiment, 3 new pigeons were exposed to a different sequence of colors, and the final stimulus was replaced in successive conditions by a novel color, a darkened key, and a restoration of the original color. The data indicated that darkening the key had a more severe, more extensive, and more persistent effect than did a mere change in color. These results suggest that it may be fruitful to conceptualize the autoshaping procedure as a special version of the block clock in which pecking is suppressed throughout the greater part of the interval by darkening the key. In the final condition, the same stimulus appeared in each of the last three portions of the interval. The rate of pecking was lower during the last two portions than when distinctive colors were presented, with the peak rate now appearing in the fifth of seven equal temporal components.

The role of the glycine modulatory site in N -methyl- d -aspartic receptor function was examined by determining the effect of the glycine site antagonist, 7-chlorokynurenic acid, on the induction of long-term potentiation at the commissural-CA1 synapse in anesthetized rats. Robust long-term potentiation of population excitatory postsynaptic potentials and population spike responses recorded extracellularly in the stratum pyramidale and in stratum radiatum of CA1 developed after high frequency stimulation (100 Hz for 1 s) of commissural fibers during continuous intrahippocampal administration of vehicle solution (0.15 M NaCl). In contrast, infusion of either 7-chlorokynurenic acid (400 μM) or of the N -methyl- d -aspartic receptor antagonist, d -2-amino-5-phosphonovaleric acid (100 μM), significantly attenuated or completely blocked the development of long-term potentiation. When 7-chlorokynurenic acid was infused together with the glycine analog, d -serine (1 mM), long-term potentiation developed that was comparable to that observed in control animals. Intrahippocampal administration of d -serine alone was associated with slightly greater magnitude of long-term potentiation than observed in control animals. Collectively, these findings establish that in intact hippocampus, activity at the glycine modulatory site is necessary for activation of the N -methyl- d -aspartic receptor complex. Furthermore, these results suggest that the glycine modulatory site may not be fully saturated in vivo , and thus can serve to regulate N -methyl- d -aspartate receptor function.

Little sodium appetite is observed when rats are deprived of dietary sodium for several days, presumably because aldosterone secretion minimizes renal sodium losses. However, the present studies indicate that when sodium deprivation is extended to 8 days, a spontaneous sodium appetite results that far exceeds urinary sodium losses during the deprivation period; indeed, adult male rats drank as much 0.5 M NaCl solution as rats ever have been reported to drink rapidly. In contrast, female rats drank much less saline after 8 days of sodium deprivation. Because of this sexual dimorphism in sodium appetite, we also studied NaCl intake in gonadectomized rats after 8 days of sodium deprivation. Both male and female gonadectomized rats drank comparable amounts of saline as intact male rats, but they consumed much less when treated with physiological amounts of estrogen during the sodium-deprivation period. These results indicate that a robust appetite for NaCl can be produced in rats by prolonged sodium deprivation and that estrogen can blunt the induced sodium appetite.

We examined in rat pups the relation between the decline of suckling and the emergence of independent ingestion, and the role of milk delivery conditional on nipple attachment in the maintenance of suckling. Fifteen-day-old litters were reared for either 5 or 10 days, and 20-day-old litters for 5 days by either a thelectomized (no nipples), ligated (nipples but no milk), or intact (nipples and milk) dam. Pups' food and water intakes were monitored daily, and their suckling, feeding, and drinking behaviors were videorecorded for 24 hr in the presence of their foster dam (Day 19 or 24) and for 24 hr in the presence of an intact, lactating dam (Day 20 or 25). There were no differences between treatment conditions with respect to either the onset or rate of increase of independent feeding or drinking. Pups reared by a thelectomized dam for 10 days displayed a pronounced, lasting depression of suckling. Twenty-five-day-old pups reared by a ligated dam displayed suckling levels comparable to those of control pups; in the presence of the ligated dam, however, their tendency to attach to a nipple was notably reduced. The implications of the findings for our understanding of the weaning process are discussed.

Maternal behavior during the weaning period (postpartum Days 14-35) was described from continuous timelapse videorecordings of Norway rat dams and their litters. Time spent nursing declined steadily after Day 20 but persisted until Day 34, about a week longer than suggested by reports of milk transfer. Most of the decline in nursing was due to progressively fewer nursing bouts per day; milk letdowns per day were consequently diminished. Although a private feeding chamber was available to the dams, they did not use this chamber to spend more time away from the pups as weaning progressed. Instead, the dams remained with the pups in the nesting chamber but devoted increasingly less time to nursing. Subtle, progressive changes in maternal behavior are closely orchestrated and coordinated with pup development.

Three experiments were conducted employing a continuous measure of conditional stimulus/unconditional stimulus (CS/US) contingencies as perceived by the subject (i.e., subjective contingency or SC). It is argued that direct measurement of relational learning, as indexed by SC, can lead to a better understanding of Pavlovian conditioning processes. The first two experiments applied this approach to a methodologic controversy, raising the debate from a procedure-based argument to testing what the subject actually learns about CS/US relationships. While the issue was not resolved, testable hypotheses for future research were generated from the data. The third experiment contrasted the contingency stimulus-stimulus (S-S) account of Pavlovian conditioning with an earlier stimulus-response (S-R) continguity-reinforcement account. In this experiment, both SC and skin resistance were measured. Evidence for the existence of both cognitive-propositional and response-learning processes in conditioning was obtained.

Two experiments were conducted to investigate the role of milk availability in the development of independent feeding and drinking in the Norway rat pup. Beginning on Day 14 postnatally, pups were exposed to different levels of milk supply by (a) changing litter size from 8 to 4 versus 12 pups (Experiment 1) or (b) limiting temporally the pups' access to a lactating dam to 8,14, or 24 hr daily (Experiment 2). Both manipulations accelerated weaning in milk-poor pups in comparison with same-age pups with relatively greater milk supplies. By adding solid food and water to their diet, early weaning pups compensated for the negative energy consequences of milk reduction and achieved premanipulation growth rates. Milk availability thus appears to affect weaning, and it is suggested that the developmental changes in the nutritive energy balance between mother and offspring contribute to the emergence of independent ingestion.

We examined the relation between milk availability and milk intake during the period in which rat pups gradually abandon milk as a food source. The amount of milk produced by rat dams does not change from postpartum Day 15 to Day 20, but decreases thereafter and completely disappears around Day 30. In contrast, the amount of milk actually obtained by pups does begin to decline between Days 15 and 20. This decline in milk intake can be attenuated by integrating 20-day-old pups into 15-day-old litters. We concluded that pups do not begin to ingest less milk because of diminishing milk supplies. Rather, the decreased tendency of mothers to nurse older pups and the diminished tendency of older pups to extract available milk, together appear to underlie the decline in pups' milk consumption. Milk supplies decline after changes in behavioral interactions and may play an instrumental role in the eventual abandonment of suckling.