Defective synaptic plasticity in a model of Coffin-Lowry syndrome is rescued by simultaneously targeting PKA and MAPK pathways.

Empirical and computational methods were combined to examine whether individual or dual-drug treatments can restore the deficit in long-term synaptic facilitation (LTF) of the Aplysia sensorimotor synapse observed in a cellular model of Coffin-Lowry syndrome (CLS). The model was produced by pharmacological inhibition of p90 ribosomal S6 kinase (RSK) activity. In this model, coapplication of an activator of the mitogen-activated protein kinase (MAPK) isoform ERK and an activator of protein kinase A (PKA) resulted in enhanced phosphorylation of RSK and enhanced LTF to a greater extent than either drug alone and also greater than their additive effects, which is termed synergism. The extent of synergism appeared to depend on another MAPK isoform, p38 MAPK. Inhibition of p38 MAPK facilitated serotonin (5-HT)-induced RSK phosphorylation, indicating that p38 MAPK inhibits activation of RSK. Inhibition of p38 MAPK combined with activation of PKA synergistically activated both ERK and RSK. Our results suggest that cellular models of disorders that affect synaptic plasticity and learning, such as CLS, may constitute a useful strategy to identify candidate drug combinations, and that combining computational models with empirical tests of model predictions can help explain synergism of drug combinations.

Quantitative description of the interactions among kinase cascades underlying long-term plasticity of Aplysia sensory neurons.

Kinases play critical roles in synaptic and neuronal changes involved in the formation of memory. However, significant gaps exist in the understanding of how interactions among kinase pathways contribute to the mechanistically distinct temporal domains of memory ranging from short-term memory to long-term memory (LTM). Activation of protein kinase A (PKA) and mitogen-activated protein kinase (MAPK)-ribosomal S6 kinase (RSK) pathways are critical for long-term enhancement of neuronal excitability (LTEE) and long-term synaptic facilitation (LTF), essential processes in memory formation. This study provides new insights into how these pathways contribute to the temporal domains of memory, using empirical and computational approaches. Empirical studies of Aplysia sensory neurons identified a positive feedforward loop in which the PKA and ERK pathways converge to regulate RSK, and a negative feedback loop in which p38 MAPK inhibits the activation of ERK and RSK. A computational model incorporated these findings to simulate the dynamics of kinase activity produced by different stimulus protocols and predict the critical roles of kinase interactions in the dynamics of these pathways. These findings may provide insights into the mechanisms underlying aberrant synaptic plasticity observed in genetic disorders such as RASopathies and Coffin-Lowry syndrome.

Unique Configurations of Compression and Truncation of Neuronal Activity Underlie l-DOPA-Induced Selection of Motor Patterns in Aplysia.

A key issue in neuroscience is understanding the ways in which neuromodulators such as dopamine modify neuronal activity to mediate selection of distinct motor patterns. We addressed this issue by applying either low or high concentrations of l-DOPA (40 or 250 µM) and then monitoring activity of up to 130 neurons simultaneously in the feeding circuitry of Aplysia using a voltage-sensitive dye (RH-155). l-DOPA selected one of two distinct buccal motor patterns (BMPs): intermediate (low l-DOPA) or bite (high l-DOPA) patterns. The selection of intermediate BMPs was associated with shortening of the second phase of the BMP (retraction), whereas the selection of bite BMPs was associated with shortening of both phases of the BMP (protraction and retraction). Selection of intermediate BMPs was also associated with truncation of individual neuron spike activity (decreased burst duration but no change in spike frequency or burst latency) in neurons active during retraction. In contrast, selection of bite BMPs was associated with compression of spike activity (decreased burst latency and duration and increased spike frequency) in neurons projecting through specific nerves, as well as increased spike frequency of protraction neurons. Finally, large-scale voltage-sensitive dye recordings delineated the spatial distribution of neurons active during BMPs and the modification of that distribution by the two concentrations of l-DOPA.

Superior long-term synaptic memory induced by combining dual pharmacological activation of PKA and ERK with an enhanced training protocol.

Developing treatment strategies to enhance memory is an important goal of neuroscience research. Activation of multiple biochemical signaling cascades, such as the protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) pathways, is necessary to induce long-term synaptic facilitation (LTF), a correlate of long-term memory (LTM). Previously, a computational model was developed which correctly predicted a novel enhanced training protocol that augmented LTF by searching for the protocol with maximal overlap of PKA and ERK activation. The present study focused on pharmacological approaches to enhance LTF. Combining an ERK activator, NSC, and a PKA activator, rolipram, enhanced LTF to a greater extent than did either drug alone. An even greater increase in LTF occurred when rolipram and NSC were combined with the Enhanced protocol. These results indicate superior memory can be achieved by enhanced protocols that take advantage of the structure and dynamics of the biochemical cascades underlying memory formation, used in conjunction with combinatorial pharmacology.

Rescue of impaired long-term facilitation at sensorimotor synapses of Aplysia following siRNA knockdown of CREB1.

Memory impairment is often associated with disrupted regulation of gene induction. For example, deficits in cAMP response element-binding protein (CREB) binding protein (CBP; an essential cofactor for activation of transcription by CREB) impair long-term synaptic plasticity and memory. Previously, we showed that small interfering RNA (siRNA)-induced knockdown of CBP in individual sensory neurons significantly reduced levels of CBP and impaired 5-HT-induced long-term facilitation (LTF) in sensorimotor cocultures from Aplysia. Moreover, computational simulations of the biochemical cascades underlying LTF successfully predicted training protocols that restored LTF following CBP knockdown. We examined whether simulations could also predict a training protocol that restores LTF impaired by siRNA-induced knockdown of the transcription factor CREB1. Simulations based on a previously described model predicted rescue protocols that were specific to CREB1 knockdown. Empirical studies demonstrated that one of these rescue protocols partially restored impaired LTF. In addition, the effectiveness of the rescue protocol was enhanced by pretreatment with rolipram, a selective cAMP phosphodiesterase inhibitor. These results provide further evidence that computational methods can help rescue disruptions in signaling cascades underlying memory formation. Moreover, the study demonstrates that the effectiveness of computationally designed training protocols can be enhanced with complementary pharmacological approaches.

Doxorubicin attenuates serotonin-induced long-term synaptic facilitation by phosphorylation of p38 mitogen-activated protein kinase.

Doxorubicin (DOX) is an anthracycline used widely for cancer chemotherapy. Its primary mode of action appears to be topoisomerase II inhibition, DNA cleavage, and free radical generation. However, in non-neuronal cells, DOX also inhibits the expression of dual-specificity phosphatases (also referred to as MAPK phosphatases) and thereby inhibits the dephosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK), two MAPK isoforms important for long-term memory (LTM) formation. Activation of these kinases by DOX in neurons, if present, could have secondary effects on cognitive functions, such as learning and memory. The present study used cultures of rat cortical neurons and sensory neurons (SNs) of Aplysia to examine the effects of DOX on levels of phosphorylated ERK (pERK) and phosphorylated p38 (p-p38) MAPK. In addition, Aplysia neurons were used to examine the effects of DOX on long-term enhanced excitability, long-term synaptic facilitation (LTF), and long-term synaptic depression (LTD). DOX treatment led to elevated levels of pERK and p-p38 MAPK in SNs and cortical neurons. In addition, it increased phosphorylation of the downstream transcriptional repressor cAMP response element-binding protein 2 in SNs. DOX treatment blocked serotonin-induced LTF and enhanced LTD induced by the neuropeptide Phe-Met-Arg-Phe-NH2. The block of LTF appeared to be attributable to overriding inhibitory effects of p-p38 MAPK, because LTF was rescued in the presence of an inhibitor (SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]) of p38 MAPK. These results suggest that acute application of DOX might impair the formation of LTM via the p38 MAPK pathway.

Computational design of enhanced learning protocols.

Learning and memory are influenced by the temporal pattern of training stimuli. However, the mechanisms that determine the effectiveness of a particular training protocol are not well understood. We tested the hypothesis that the efficacy of a protocol is determined in part by interactions among biochemical cascades that underlie learning and memory. Previous findings suggest that the protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) cascades are necessary to induce long-term synaptic facilitation (LTF) in Aplysia, a neuronal correlate of memory. We developed a computational model of the PKA and ERK cascades and used it to identify a training protocol that maximized PKA and ERK interactions. In vitro studies confirmed that the protocol enhanced LTF. Moreover, the protocol enhanced the levels of phosphorylation of the transcription factor CREB1. Behavioral training confirmed that long-term memory also was enhanced by the protocol. These results illustrate the feasibility of using computational models to design training protocols that improve memory.

The ubiquitin-proteasome system is necessary for long-term synaptic depression in Aplysia.

The neuropeptide Phe-Met-Arg-Phe-NH(2) (FMRFa) can induce transcription-dependent long-term synaptic depression (LTD) in Aplysia sensorimotor synapses. We investigated the role of the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to presynaptic inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by CREB2, which is generally regarded as a transcription repressor. Binding of CREB2 to the promoter region of ap-uch was accompanied by histone hyperacetylation, suggesting that CREB2 cannot only inhibit but also promote gene expression. CREB2 was phosphorylated after FMRFa, and blocking phospho-CREB2 blocked LTD. In addition to changes in the expression of ap-uch, the synaptic vesicle-associated protein synapsin was downregulated in LTD in a proteasome-dependent manner. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions.

Molecular mechanisms underlying a cellular analog of operant reward learning.

Operant conditioning is a ubiquitous but mechanistically poorly understood form of associative learning in which an animal learns the consequences of its behavior. Using a single-cell analog of operant conditioning in neuron B51 of Aplysia, we examined second-messenger pathways engaged by activity and reward and how they may provide a biochemical association underlying operant learning. Conditioning was blocked by Rp-cAMP, a peptide inhibitor of PKA, a PKC inhibitor, and by expressing a dominant-negative isoform of Ca2+-dependent PKC (apl-I). Thus, both PKA and PKC were necessary for operant conditioning. Injection of cAMP into B51 mimicked the effects of operant conditioning. Activation of PKC also mimicked conditioning but was dependent on both cAMP and PKA, suggesting that PKC acted at some point upstream of PKA activation. Our results demonstrate how these molecules can interact to mediate operant conditioning in an individual neuron important for the expression of the conditioned behavior.

Changes in neuronal excitability serve as a mechanism of long-term memory for operant conditioning.

Learning can lead to changes in the intrinsic excitability of neurons. However, the extent to which these changes persist and the role they have in the expression of memory remain unclear. We found that in vitro analogs of operant conditioning produced a long-term (24 h) increase in the excitability of an identified neuron (B51) that is critical for the expression of feeding in Aplysia. This increase in excitability, which was cAMP dependent, contributed to the associative modification of the feeding circuitry, providing a mechanism for long-term memory.

Bistable MAP kinase activity: a plausible mechanism contributing to maintenance of late long-term potentiation.

Bistability of MAP kinase (MAPK) activity has been suggested to contribute to several cellular processes, including differentiation and long-term synaptic potentiation. A recent model (Markevich NI, Hoek JB, Kholodenko BN. J Cell Biol 164: 353-359, 2004) predicts bistability due to interactions of the kinases and phosphatases in the MAPK pathway, without feedback from MAPK to earlier reactions. Using this model and enzyme concentrations appropriate for neurons, we simulated bistable MAPK activity, but bistability was present only within a relatively narrow range of activity of Raf, the first pathway kinase. Stochastic fluctuations in molecule numbers eliminated bistability for small molecule numbers, such as are expected in the volume of a dendritic spine. However, positive-feedback loops have been posited from MAPK up to Raf activation. One proposed loop in which MAPK directly activates Raf was incorporated into the model. We found that such feedback greatly enhanced the robustness of both stable states of MAPK activity to stochastic fluctuations and to parameter variations. Bistability was robust for molecule numbers plausible for a dendritic spine volume. The upper state of MAPK activity was resistant to inhibition of MEK activation for >1 h, which suggests that inhibitor experiments have not sufficed to rule out a role for persistent MAPK activity in the maintenance of long-term potentiation (LTP). These simulations suggest that persistent MAPK activity and consequent upregulation of translation may contribute to LTP maintenance and to long-term memory. Experiments using a fluorescent MAPK substrate may further test this hypothesis.

Synapsin regulates Basal synaptic strength, synaptic depression, and serotonin-induced facilitation of sensorimotor synapses in Aplysia.

Synapsin is a synaptic vesicle-associated protein implicated in the regulation of vesicle trafficking and transmitter release, but its role in heterosynaptic plasticity remains elusive. Moreover, contradictory results have obscured the contribution of synapsin to homosynaptic plasticity. We previously reported that the neuromodulator serotonin (5-HT) led to the phosphorylation and redistribution of Aplysia synapsin, suggesting that synapsin may be a good candidate for the regulation of vesicle mobilization underlying the short-term synaptic plasticity induced by 5-HT. This study examined the role of synapsin in homosynaptic and heterosynaptic plasticity. Overexpression of synapsin reduced basal transmission and enhanced homosynaptic depression. Although synapsin did not affect spontaneous recovery from depression, it potentiated 5-HT-induced dedepression. Computational analysis showed that the effects of synapsin on plasticity could be adequately simulated by altering the rate of Ca(2+)-dependent vesicle mobilization, supporting the involvement of synapsin not only in homosynaptic but also in heterosynaptic forms of plasticity by regulating vesicle mobilization.

Transforming growth factor-beta2 modulates synaptic efficacy and plasticity and induces phosphorylation of CREB in hippocampal neurons.

Transforming growth factor-betas (TGF-betas) are widely expressed and play roles as multifunctional growth factors and regulators of key events in development, disease, and repair. However, it is not known whether TGF-betas affect the plasticity of hippocampal neurons. As a first step to address this issue, we examined whether TGF-beta2 modulated the electrophysiological and biochemical properties of cultured hippocampal neurons. We found that prolonged 24 h treatment with TGF-beta2 induced facilitation of evoked postsynaptic currents (ePSCs). This facilitation was associated with a decrease in short-term synaptic depression of ePSCs and increases in both the amplitude and frequency of spontaneous miniature postsynaptic currents (mPSCs). The long-term changes of ePSCs and mPSCs may be associated with cAMP response element-binding protein (CREB), which has been previously implicated in long-term potentiation. Immunofluorescence techniques and Western blot analysis both revealed that TGF-beta2 enhanced the phosphorylation of CREB. Together, these results suggest that TGF-beta2 may play a role in the cascade of events underlying long-term synaptic facilitation in hippocampus, and that CREB may be an important mediator of these effects.

A model of the roles of essential kinases in the induction and expression of late long-term potentiation.

The induction of late long-term potentiation (L-LTP) involves complex interactions among second-messenger cascades. To gain insights into these interactions, a mathematical model was developed for L-LTP induction in the CA1 region of the hippocampus. The differential equation-based model represents actions of protein kinase A (PKA), MAP kinase (MAPK), and CaM kinase II (CAMKII) in the vicinity of the synapse, and activation of transcription by CaM kinase IV (CAMKIV) and MAPK. L-LTP is represented by increases in a synaptic weight. Simulations suggest that steep, supralinear stimulus-response relationships between stimuli (e.g., elevations in [Ca(2+)]) and kinase activation are essential for translating brief stimuli into long-lasting gene activation and synaptic weight increases. Convergence of multiple kinase activities to induce L-LTP helps to generate a threshold whereby the amount of L-LTP varies steeply with the number of brief (tetanic) electrical stimuli. The model simulates tetanic, -burst, pairing-induced, and chemical L-LTP, as well as L-LTP due to synaptic tagging. The model also simulates inhibition of L-LTP by inhibition of MAPK, CAMKII, PKA, or CAMKIV. The model predicts results of experiments to delineate mechanisms underlying L-LTP induction and expression. For example, the cAMP antagonist RpcAMPs, which inhibits L-LTP induction, is predicted to inhibit ERK activation. The model also appears useful to clarify similarities and differences between hippocampal L-LTP and long-term synaptic strengthening in other systems.

Bifurcation and singularity analysis of a molecular network for the induction of long-term memory.

Withdrawal reflexes of the mollusk Aplysia exhibit sensitization, a simple form of long-term memory (LTM). Sensitization is due, in part, to long-term facilitation (LTF) of sensorimotor neuron synapses. LTF is induced by the modulatory actions of serotonin (5-HT). Pettigrew et al. developed a computational model of the nonlinear intracellular signaling and gene network that underlies the induction of 5-HT-induced LTF. The model simulated empirical observations that repeated applications of 5-HT induce persistent activation of protein kinase A (PKA) and that this persistent activation requires a suprathreshold exposure of 5-HT. This study extends the analysis of the Pettigrew model by applying bifurcation analysis, singularity theory, and numerical simulation. Using singularity theory, classification diagrams of parameter space were constructed, identifying regions with qualitatively different steady-state behaviors. The graphical representation of these regions illustrates the robustness of these regions to changes in model parameters. Because persistent protein kinase A (PKA) activity correlates with Aplysia LTM, the analysis focuses on a positive feedback loop in the model that tends to maintain PKA activity. In this loop, PKA phosphorylates a transcription factor (TF-1), thereby increasing the expression of an ubiquitin hydrolase (Ap-Uch). Ap-Uch then acts to increase PKA activity, closing the loop. This positive feedback loop manifests multiple, coexisting steady states, or multiplicity, which provides a mechanism for a bistable switch in PKA activity. After the removal of 5-HT, the PKA activity either returns to its basal level (reversible switch) or remains at a high level (irreversible switch). Such an irreversible switch might be a mechanism that contributes to the persistence of LTM. The classification diagrams also identify parameters and processes that might be manipulated, perhaps pharmacologically, to enhance the induction of memory. Rational drug design, to affect complex processes such as memory formation, can benefit from this type of analysis.

An aplysia dopamine1-like receptor: molecular and functional characterization.

In Aplysia, the neurotransmitter dopamine is involved in the regulation of various physiological processes and motor functions, like feeding behaviour, and in the siphon-gill withdrawal reflex. In this paper, we report the characterization of the first Aplysia D1-like dopamine receptor (Apdop1) mainly expressed in the CNS, heart and buccal mass. Following expression of the Apdop1 receptor in HEK293 cells, a higher level of cAMP was observed in the absence of the receptor ligand, showing that Apdop1 is constitutively active. This activity was blocked by the inverse agonist flupentixol. Application of dopamine (EC50 of 35 nm) or serotonin (EC50 of 36 microm) to Apdop1-transfected HEK293 cells further increased the level of cAMP, suggesting that the receptor is linked to the stimulatory Gs protein pathway. When expressed in cultured sensory neurons, Apdop1 immunoreactivity was observed in the cell body and neurites. Control sensory neurons responded to dopamine with a decrease in excitability mediated by a pertusis toxin-sensitive G protein. Expression of Apdop1 produced an increase in hyperpolarization in the absence of agonist and an increase in membrane excitability following stimulation by dopamine. In the presence of pertussis toxin to inhibit the Gi protein inhibitory pathway responsible for decrease in excitability mechanism, Stimulation of membrane excitability was observed. Apdop1 sensitivity to dopamine makes it a potential modulator of operant conditioning procedure.

Teaching basic principles of neuroscience with computer simulations.

It is generally believed that students learn best through activities that require their direct participation. By using simulations as a tool for learning neuroscience, students are directly engaged in the activity and obtain immediate feedback and reinforcement. This paper describes a series of biophysical models and computer simulations that can be used by educators and students to explore a variety of basic principles in neuroscience. The paper also suggests 'virtual laboratory' exercises that students may conduct to further examine biophysical processes underlying neural function. First, the Hodgkin and Huxley (HH) model is presented. The HH model is used to illustrate the action potential, threshold phenomena, and nonlinear dynamical properties of neurons (e.g., oscillations, postinhibitory rebound excitation). Second, the Morris-Lecar (ML) model is presented. The ML model is used to develop a model of a bursting neuron and to illustrate modulation of neuronal activity by intracellular ions. Lastly, principles of synaptic transmission are presented in small neural networks, which illustrate oscillatory behavior, excitatory and inhibitory postsynaptic potentials, and temporal summation.

cAMP-response elements in Aplysia creb1, creb2, and Ap-uch promoters: implications for feedback loops modulating long term memory.

The Aplysia genes encoding for cAMP-response element-binding protein 1 (CREB1), CREB2, and ubiquitin C-terminal hydrolase (Ap-uch) have been implicated in the formation of long term memory. However, nothing is known about the promoter regions of these genes or the transcription factors that regulate them. We cloned the promoter regions of creb1, creb2, and Ap-uch and identified a canonical cAMP-response element (CRE) in the promoter region of creb1. Variants of the canonical CRE were identified in all three promoters. TATA boxes and C/EBP-binding motifs are also present in the promoter regions of these genes. Promoter immunoprecipitation assays and chromatin immunoprecipitation assays indicated that CREB1 and CREB2 bind to the promoter regions of creb1 and creb2, suggesting that feedback loops modulate the formation of long term memory. In a positive feedback loop, phosphorylated CREB1 might induce its own gene via CREs. In support of this suggestion, treatment with serotonin enhanced binding of CREB1 to its promoter region and increased mRNA levels of creb1. Levels of Ap-uch mRNA also increased in response to serotonin; however, binding of CREB1 or CREB2 to the promoter region of Ap-uch was not detected. The finding that the promoter region of creb2 has a CRE raises the intriguing possibility that its expression is regulated by CREB1 and/or CREB2. CREB2 may repress its own gene, forming a negative feedback loop, and CREB2 up-regulation via CREB1 may limit the activity of the CREB1-mediated positive feedback loop.

Dynamic properties of regulatory motifs associated with induction of three temporal domains of memory in aplysia.

A model was developed to examine dynamical properties of regulatory motifs correlated with different temporal domains of memory. The model represents short-, intermediate-, and long-term phases of protein kinase A (PKA) activation, which appear related to corresponding phases of facilitation of the Aplysia sensorimotor synapse. The model also represents phosphorylation of the transcription factor CREB1 by PKA and consequent induction of the immediate-early gene Aplysia ubiquitin hydrolase (Ap-uch), which is essential for long-term synaptic facilitation (LTF). Simulations suggest mechanisms responsible for differing profiles of synaptic facilitation following massed vs. spaced exposures to 5-HT, and suggest a novel regulatory motif (gated positive feedback) is important for LTF. Simulations suggest zero-order ultrasensitivity may underlie a requirement of a threshold number of exposures to 5-HT for LTF induction. The model makes predictions for the dynamics of PKA activation and Ap-uch induction when MAP kinase is activated, or when repression of Ap-uch is relieved by inhibiting the transcription factor CREB2. This model may therefore be useful for understanding processes underlying memory formation in Aplysia and other systems.

Coregulation of glutamate uptake and long-term sensitization in Aplysia.

In Aplysia, long-term facilitation (LTF) at sensorimotor synapses of the pleural-pedal ganglia is mediated by an increase in the release of a neurotransmitter, which appears to be glutamate. Glutamate uptake also is increased in sensory neurons 24 hr after the induction of long-term sensitization (Levenson et al., 2000b). The present study investigated whether the same signaling pathways were involved in the long-term increase in glutamate uptake as in the induction of LTF. Thus, roles for cAMP, PKA (cAMP-dependent protein kinase), MAPK (mitogen-activated protein kinase), and tyrosine kinase in the regulation of glutamate uptake were tested. We found that 5-HT increased cAMP and activated PKA in sensory neurons. Exposure of pleural-pedal ganglia to analogs of cAMP or forskolin increased glutamate uptake 24 hr after treatments. Inhibitors of PKA (KT5720), MAPK (U0126 and PD98059), and tyrosine kinase (genistein) blocked the long-term increase in glutamate uptake produced by 5-HT. In addition, bpV, a tyrosine phosphatase inhibitor, facilitated the ability of subthreshold levels of 5-HT to increase glutamate uptake. Inhibition of PKC, which is not involved in LTF, had no effect on the long-term increase in glutamate uptake produced by 5-HT. Furthermore, activation of PKC by phorbol-12,13-dibutyrate did not produce long-term changes in glutamate uptake. The results demonstrate that the same constellation of second messengers and kinases is involved in the long-term regulation of both glutamate release and glutamate uptake. These similarities in signaling pathways suggest that regulation of glutamate release and uptake during formation of long-term memory are coordinated through coregulation of these two processes.