Danger and distress: Parabrachial-extended amygdala circuits.

Our understanding of the role of the parabrachial nucleus (PBN) has evolved as technology has advanced, in part due to cell-specific studies and complex behavioral assays. This is reflected in the heterogeneous neuronal populations within the PBN to the extended amygdala (EA) circuits which encompass the bed nucleus of the stria terminalis (BNST) and central amygdala (CeA) circuitry, as they differentially modulate aspects of behavior in response to diverse threat-like contexts necessary for survival. Here we review how the PBN→CeA and PBN→BNST pathways differentially modulate fear-like behavior, innate and conditioned, through unique changes in neurotransmission in response to stress-inducing contexts. Furthermore, we hypothesize how in specific instances the PBN→CeA and PBN→BNST circuits are redundant and in part intertwined with their respective reciprocal projections. By deconstructing the interoceptive and exteroceptive components of affect- and stress related behavioral paradigms, evidence suggests that the PBN→CeA circuit modulates innate response to physical stimuli and fear conditioning. Conversely, the PBN→BNST circuit modulates distress-like stress in unpredictable contexts. Thereby, the PBN provides a pathway for alarming interoceptive and exteroceptive stimuli to be processed and relayed to the EA to induce stress-relevant affect. Additionally, we provide a framework for future studies to detail the cell-type specific intricacies of PBN→EA circuits in mediating behavioral responses to threats, and the relevance of the PBN in drug-use as it relates to threat and negative reinforcement. This article is part of the special Issue on 'Neurocircuitry Modulating Drug and Alcohol Abuse'.

Cocaine Augments Dopamine Mediated Inhibition of Neuronal Activity in the Dorsal Bed Nucleus of the Stria Terminalis.

The dorsal region of the bed nucleus of the stria terminalis (dBNST) receives substantial dopaminergic input which overlaps with norepinephrine input implicated in stress responses. Using ex vivo fast scan cyclic voltammetry in male C57BL6 mouse brain slices, we demonstrate that electrically stimulated dBNST catecholamine signals are of substantially lower magnitude and have slower uptake rates compared to caudate signals. Dopamine terminal autoreceptor activation inhibited roughly half of the catecholamine transient, and noradrenergic autoreceptor activation produced an ∼30% inhibition. Dopamine transporter blockade with either cocaine or GBR12909 significantly augmented catecholamine signal duration. We optogenetically targeted dopamine terminals in the dBNST of transgenic (TH:Cre) mice of either sex and, using ex vivo whole-cell electrophysiology, we demonstrate that optically stimulated dopamine release induces slow outward membrane currents and an associated hyperpolarization response in a subset of dBNST neurons. These cellular responses had a similar temporal profile to dopamine release, were significantly reduced by the D2/D3 receptor antagonist raclopride, and were potentiated by cocaine. Using in vivo fiber photometry in male C57BL6 mice during training sessions for cocaine conditioned place preference, we show that acute cocaine administration results in a significant inhibition of calcium transient activity in dBNST neurons compared to saline administration. These data provide evidence for a mechanism of dopamine-mediated cellular inhibition in the dBNST and demonstrate that cocaine augments this inhibition while also decreasing net activity in the dBNST in a drug reinforcement paradigm.SIGNIFICANCE STATEMENTThe dorsal bed nucleus of the stria terminalis (dBNST) is a region highly implicated in mediating stress responses, however, the dBNST also receives dopaminergic inputs from classically defined drug reward pathways. Here we used various techniques to demonstrate that dopamine signaling within the dorsal BNST region has inhibitory effects on population activity. We show that cocaine, an abused psychostimulant, augments both catecholamine release and dopamine-mediated cellular inhibition in this region. We also demonstrate that cocaine administration reduces population activity in the dBNST, in vivo Together these data support a mechanism of dopamine-mediated inhibition within the dBNST, providing a means by which drug-induced elevations in dopamine signaling may inhibit dBNST activity to promote drug reward.

BNST transient activity associates with approach behavior in a stressful environment and is modulated by the parabrachial nucleus.

Studies demonstrate a role for the bed nucleus of the stria terminalis (BNST) in modulating affective behavior and stress-reward integration. To explore the dynamic nature of in vivo BNST activity associated with anxiety-like behavior in a stress-inducing context, we utilized fiber photometry and detected BNST calcium transients in mice during the novelty-suppressed feeding task (NSFT). Phasic BNST activity emerged time-locked to novel object or food pellet approach during NSFT. The parabrachial nucleus (PBN) has a large input to the BNST and is thought to function as a danger signal, in arousal responses and in feeding behavior. To explore a potential role for the PBN as a contributor to BNST activity in NSFT, we investigated whether chemogenetic regulation of PBN activity altered the dynamic BNST response synchronized to NSFT approach behavior. We found that activation of the hM3D(Gq) DREADD in the PBN enhanced the observed transient signal in the BNST synchronized to the consummatory food approach, and was associated at the behavioral level with increased latency to consume food. Because the PBN has multiple efferent pathways, we next used a transsynaptic anterograde AAV-based strategy to express hM3D(Gq) specifically in PBN-innervated BNST (BNSTPBN) neurons in male and female mice. Activation of hM3D(Gq) in these BNSTPBN neurons increased latency to consume food in female, but not male mice. To further explore the population of BNST neurons contributing to phasic BNST activity associated with NSFT, we turned to PKCδ neurons in BNST. BNST(PKCδ) neurons are implicated in stress and food-related behavior, and we previously found that the expression of this kinase is regulated in the BNST by stress in a sex-dependent manner. Here, we demonstrate close apposition of CGRP, a marker of PBN terminals, adjacent to BNST(PKCδ) cells. Finally, we find that PKCδ-expressing BNST cells exhibit a large transient signal synchronized to the consummatory food approach similar to that seen with bulk BNST activity measures. Taken together these data demonstrate phasic BNST activity at a global and cell-specific level that is driven in part by PBN activity at the time of NSFT consummatory approach behavior.

Preclinical voluntary drinking models for alcohol abstinence-induced affective disturbances in mice.

Negative reinforcement is widely thought to play an important role in chronic alcohol-use disorders (AUDs), and high comorbidity between AUDs and affective disorders highlights the importance of investigating this relationship. Prominent models posit that repeated cycles of alcohol (ethanol, EtOH) exposure and withdrawal produce circuit adaptations in the central nervous system that drive a transition from positive- to negative reinforcement-based alcohol seeking. Evidence supporting this theory has accumulated in large part using forced EtOH administration models, such as chronic intragastric gavage and chronic vapor inhalation. However, recent studies utilizing simple voluntary EtOH delivery systems show that forced abstinence from EtOH intake administered by the animal itself can produce evolving and significant affective disturbances, particularly in female C57BL/6J mice. Here, we highlight these recent studies to support the idea that voluntary EtOH administration in mouse models, as well as a protracted abstinence period and less commonly used behavioral tasks, could unveil affective disturbances during abstinence that have remained elusive using high dosage forced EtOH administration paradigms.

Knockdown of BNST GluN2B-containing NMDA receptors mimics the actions of ketamine on novelty-induced hypophagia.

Administration of a single low dose of the N-methyl-D-aspartate (NMDA) receptor antagonist ketamine has been demonstrated to elicit long-lasting antidepressant effects in humans with depression, as well as in rodent models of depression. Although pharmacological studies have implicated the GluN2B subunit of the NMDA receptor in these effects, drugs targeting this subunit have off-target actions, and systemic administration of these compounds does not allow for delineation of specific brain regions involved. In this study, we assessed the role of GluN2B in the bed nucleus of the stria terminalis (BNST) in novelty-induced hypophagia (NIH) in mice. First, we verified that ketamine, as well as the GluN2B antagonist Ro25-6981, decreased the latency to consume food in a novel environment in a version of the NIH test. We then hypothesized that GluN2B-containing receptors within the BNST may be a target of systemic ketamine and contribute to behavioral effects. Through the combination of a GluN2B-floxed mouse line and stereotaxic delivery of lentiviral Cre recombinase, we found that targeted knockdown of this subunit within the BNST mimicked the reduction in affective behavior observed with systemic ketamine or Ro25-6981 in the NIH test. These data suggest a role for GluN2B-containing NMDARs within the BNST in the affective effects of systemic ketamine.

Developmental changes in the acute ethanol sensitivity of glutamatergic and GABAergic transmission in the BNST.

Glutamatergic and GABAergic transmission undergo significant changes during adolescence. Receptors for both of these transmitters (NMDAR, and GABAA) are known to be key targets for the acute effects of ethanol in adults. The current study set out to investigate the acute effects of ethanol on both NMDAR-mediated excitatory transmission and GABAergic inhibitory transmission within the bed nucleus of the stria terminalis (BNST) across age. The BNST is an area of the brain implicated in the negative reinforcing properties associated with alcohol dependence, and the BNST plays a critical role in stress-induced relapse. Therefore, assessing the developmental regulation of ethanol sensitivity in this key brain region is important to understanding the progression of ethanol dependence. To do this, whole-cell recordings of isolated NMDAR-evoked excitatory postsynaptic currents (eEPSCs) or evoked GABAergic inhibitory postsynaptic currents (eIPSCs) were performed on BNST neurons in slices from 4- or 8-week-old male C57BL/6J mice. Ethanol (50 mm) produced greater inhibition of NMDAR-eEPSCs in adolescent mice than in adult mice. This enhanced sensitivity in adolescence was not a result of shifts in function of the GluN2B subunit of the NMDAR, measured by Ro25-6981 inhibition and decay kinetics measured across age. Adolescent mice also exhibited greater ethanol sensitivity of GABAergic transmission, as ethanol (50 mm) enhanced eIPSCs in the BNST of adolescent but not adult mice. Collectively, this work illustrates that a moderate dose of ethanol produces greater inhibition of transmission in the BNST (through greater excitatory inhibition and enhancement of inhibitory transmission) in adolescents compared to adults. Given the role of the BNST in alcohol dependence, these developmental changes in acute ethanol sensitivity could accelerate neuroadaptations that result from chronic ethanol use during the critical period of adolescence.

Absence of Ca2+-stimulated adenylyl cyclases leads to reduced synaptic plasticity and impaired experience-dependent fear memory.

Ca(2+)-stimulated adenylyl cyclase (AC) 1 and 8 are two genes that have been shown to play critical roles in fear memory. AC1 and AC8 couple neuronal activity and intracellular Ca(2+) increases to the production of cyclic adenosine monophosphate and are localized synaptically, suggesting that Ca(2+)-stimulated ACs may modulate synaptic plasticity. Here, we first established that Ca(2+)-stimulated ACs modulate protein markers of synaptic activity at baseline and after learning. Primary hippocampal cell cultures showed that AC1/AC8 double-knockout (DKO) mice have reduced SV2, a synaptic vesicle protein, abundance along their dendritic processes, and this reduction can be rescued through lentivirus delivery of AC8 to the DKO cells. Additionally, phospho-synapsin, a protein implicated in the regulation of neurotransmitter release at the synapse, is decreased in vivo 1 h after conditioned fear (CF) training in DKO mice. Importantly, additional experiments showed that long-term potentiation deficits present in DKO mice are rescued by acutely replacing AC8 in the forebrain, further supporting the idea that Ca(2+)-stimulated AC activity is a crucial modulator of synaptic plasticity. Previous studies have demonstrated that memory is continually modulated by gene-environment interactions. The last set of experiments evaluated the effects of knocking out AC1 and AC8 genes on experience-dependent changes in CF memory. We showed that the strength of CF memory in wild-type mice is determined by previous environment, minimal or enriched, whereas memory in DKO mice is unaffected. Thus, overall these results show that AC1 and AC8 modulate markers of synaptic activity and help integrate environmental information to modulate fear memory.

Evidence for a causal association for HPV in head and neck cancers.

Current data have now attributed a viral etiology and causality of Human papillomavirus (HPV). Epidemiological analysis of the last decade demonstrates a rapid increase of HPV-associated HNSCC. Genomic detection of HPV DNA in the nuclei of certain oro-pharyngeal cancer cells gives strong evidence of a viral etiology in HNSCC. Non-smokers, non-drinkers, and a sexual debut at a younger age and other sexual risk factors have an increased risk of HPV-positive oropharyngeal cancer. Sexual transmission is considered to play a causal role. In contrast to HPV-negative HNSCC most studies reveal a favorable prognosis for HPV-positive tumors. There is evidence of alterations in the p53 pathway through expression of E6 oncogene with subsequent induction of tumor cell proliferation. Synergies between viral oncogenes and other carcinogens are hypothesized. HPV alone appears to be insufficient as the sole cause of HNSCC; this may explain the long latency period between HPV infection and cancer development. There is now sufficient evidence for a causal role for HPV in HNSCC. As in cervical cancer, HPV requires oncogenes and co-factors for tumor development. Thus, inhibition or loss of such co-factors may lead to tumor regression. The vast amounts of epidemiological, molecular pathological and in vitro experimental data are consistent with the hypothesis that HPV does indeed have a causal role. We await final validation from animal experimentation in which regression of HPV-positive tumors will follow from loss or inhibition of E6 and E7.

alpha2A-adrenergic receptors heterosynaptically regulate glutamatergic transmission in the bed nucleus of the stria terminalis.

Stress is a major driving force in reinstatement of drug-seeking behavior. The bed nucleus of the stria terminalis (BNST) has been identified as a key brain region in this behavior, and receives a dense input of the stress-neurotransmitter norepinephrine through the ventral noradrenergic bundle. Activation of alpha(2)-adrenergic receptors (alpha(2)-ARs) in the BNST blocks stress-induced reinstatement of drug-seeking, indicating a potentially important role for these receptors. Currently, it is unclear how alpha(2)-AR agonists elicit this behavioral action, or through which alpha(2)-AR subtype. Activation of alpha(2)-ARs decreases glutamatergic transmission in the BNST, an effect which is nearly absent in the alpha(2A)-AR knockout mouse. Here, we take advantage of a knock-in mouse in which a hemagglutinin-tagged alpha(2A)-AR was inserted into the endogenous locus, along with the alpha(2A)-AR selective agonist guanfacine, to further study the role of the alpha(2A)-AR subtype in modulation of neurotransmission in the BNST. Using immunohistochemistry, we find that alpha(2A)-ARs are highly expressed in the BNST, and that this expression is more similar in distribution to the vesicular glutamate transporters than to either norepinephrine transporter or tyrosine hydroxylase positive terminals. Using whole cell patch-clamp recordings, we show that guanfacine causes a depression of evoked excitatory and, to a more limited extent, inhibitory fast synaptic transmission. In total, these data support a prominent heterosynaptic role for alpha(2A)-ARs in modulating fast synaptic transmission in the BNST.

An increase in DNA double-strand breaks, induced by Ku70 depletion, is associated with human papillomavirus 16 episome loss and de novo viral integration events.

Integration of human papillomavirus type 16 (HPV16) is a common event in cervical carcinogenesis, although mechanisms of integration are poorly understood. We have tested the hypothesis that an increased number of DNA double-strand breaks (DSBs) affect HPV16 episome maintenance and integration in cervical keratinocytes. Increased DSBs were generated over prolonged periods of up to 50 population doublings in the unique polyclonal cervical keratinocyte cell line W12, which stably maintains HPV16 episomes. This was achieved using repeated treatments with short interfering RNA to obtain sustained depletion of Ku70, a key mediator of DNA non-homologous end joining. An increase in DSBs was seen shortly after commencement of Ku70 depletion. Continuous depletion was reproducibly associated with loss of HPV16 episomes and also with a new viral integration event, which was rapidly selected in outgrowing W12 cells. Despite the prolonged presence of DSBs, high-level chromosomal instability (detected by marked changes in genomic copy number) was not observed until cells containing the new integrant were almost fully selected, with no evidence of such chromosomal instability prior to integration. Our data show that increased DNA DSBs are associated with HPV16 episomal loss and integration in cervical keratinocytes. We found no evidence to support the notion that major chromosomal instability precedes HPV16 integration, although such instability is an important consequence of the integration event.

Gain and overexpression of the oncostatin M receptor occur frequently in cervical squamous cell carcinoma and are associated with adverse clinical outcome.

For many oncogenes, increased expression resulting from copy number gain confers a selective advantage to cells that consequently make up the tumour bulk. To identify oncogenes of potential biological significance in cervical squamous cell carcinoma (SCC), 36 primary samples and ten cell lines were screened by array comparative genomic hybridization (CGH). The most commonly occurring regions of copy number gain that also showed amplification were 5p15.2-14.3 (59%), 5p13.3 (65%), and 5p13.2-13.1 (63%). Gene copy numbers were significantly associated with expression levels for three candidate oncogenes at these loci: OSMR (oncostatin M receptor) (p=0.03), PDZK3 (PDZ domain containing protein 3) (p=0.04), and TRIO (triple functional domain) (p=0.03). Further examination by fluorescence in situ hybridization on a tissue microarray of 110 primary cervical SCC samples revealed copy number gain frequencies of 60.9%, 57.3%, and 54.5% for OSMR, PDZK3, and TRIO, respectively, with OSMR adversely influencing overall patient survival independently of tumour stage (p=0.046). By array CGH, copy number gain of OSMR was not seen in any of 40 microdissected precursor cervical squamous intraepithelial lesions (SILs). Moreover, global mRNA expression analysis, using Affymetrix U133A 2.0 Arrays, showed no overexpression of OSMR in SILs, suggesting that OSMR gain and overexpression are relatively late steps in cervical carcinogenesis. In the cervical SCC cell lines CaSki and SW756, exogenous OSM activated downstream-signalling elements of OSMR including STAT3, p44/42 MAPK, and S6 ribosomal protein, and induced transcription of the angiogenic factor VEGF, effects that were reduced by OSMR depletion using RNA interference. We conclude that copy number gain of OSMR is frequently found in cervical SCC and is associated with adverse clinical outcome. As well as being a potential prognostic marker, OSMR is a candidate cell surface therapeutic target.

Global microRNA profiles in cervical squamous cell carcinoma depend on Drosha expression levels.

Gain of chromosome 5p is seen in over 50% of advanced cervical squamous cell carcinomas (SCCs), although the genes responsible for the selective advantage provided by this abnormality are poorly understood. In the W12 cervical carcinogenesis model, we observed that 5p gain was rapidly selected over approximately 15 population doublings and was associated with the acquisition of a growth advantage and invasiveness. The most significantly upregulated transcript following 5p gain was the microRNA (miRNA) processor Drosha. In clinically progressed cervical SCC, Drosha copy-number gain was seen in 21/36 clinical samples and 8/10 cell lines and there was a significant association between Drosha transcript levels and copy-number gain. Other genes in the miRNA processing pathway, DGCR8, XPO5 and Dicer, showed infrequent copy-number gain and over-expression. Drosha copy-number and expression were not elevated in pre-malignant cervical squamous intraepithelial lesions. Importantly, global miRNA profiling showed that Drosha over-expression in cervical SCC appears to be of functional significance. Unsupervised principal component analysis of a mixed panel of cervical SCC cell lines and clinical specimens showed clear separation according to Drosha over-expression. miRNAs most significantly associated with Drosha over-expression are implicated in carcinogenesis in other tissues, suggesting that they regulate fundamental processes in neoplastic progression. Our evidence suggests that copy-number driven over-expression of Drosha and consequent changes in miRNAs are likely to be important contributors to the selective advantage provided by 5p gain in cervical neoplastic progression.

Plasticity and behavior: new genetic techniques to address multiple forms and functions.

As the best-studied form of vertebrate synaptic plasticity, NMDA-receptor dependent long-term potentiation (NMDAR-LTP) has long been considered a leading candidate for a cellular locus for some aspects of learning and memory. However, assigning a specific role for this form of plasticity in learning and memory has proven surprisingly difficult. Two issues have contributed to this difficulty. First, a large number of molecules have been shown to in some way mediate or modulate not only NMDAR-LTP but also many forms of plasticity. Indeed, it is increasingly clear that multiple induction and maintenance mechanisms for plasticity exist, often at the same synapse. Second, linking cellular events to behavioral function has been hindered by a lack of sufficiently precise tools. In this review, we will discuss some of the proposed mechanisms of induction and maintenance of changes in synaptic efficacy and their regulation in the context of an attempt to understand their roles in animal behavior. Further, we will discuss recently developed genetic techniques, specifically, inducible transgenic models, which now allow more precise manipulations in the study of the roles plasticity plays in learning and memory.

Inducible and reversible enhancement of learning, memory, and long-term potentiation by genetic inhibition of calcineurin.

The threshold for hippocampal-dependent synaptic plasticity and memory storage is thought to be determined by the balance between protein phosphorylation and dephosphorylation mediated by the kinase PKA and the phosphatase calcineurin. To establish whether endogenous calcineurin acts as an inhibitory constraint in this balance, we examined the effect of genetically inhibiting calcineurin on plasticity and memory. Using the doxycycline-dependent rtTA system to express a calcineurin inhibitor reversibly in the mouse brain, we find that the transient reduction of calcineurin activity facilitates LTP in vitro and in vivo. This facilitation is PKA dependent and persists over several days in vivo. It is accompanied by enhanced learning and strengthened short- and long-term memory in several hippocampal-dependent spatial and nonspatial tasks. The LTP and memory improvements are reversed fully by suppression of transgene expression. These results demonstrate that endogenous calcineurin constrains LTP and memory.

Tethered flight activity of Nephotettix virescens (Hemiptera: cicadellidae) in the Philippines.

The flight potential of Nephotettix virescens (Distant), the most important vector of rice tungro virus disease, was assessed using tethered flight techniques. Most individuals tested were not willing to fly in response to stimulation, or flew for very short times. A small proportion of leafhoppers flew for long periods and one female flew for almost 7 h, indicating the potential for long distance dispersal of insects and inoculum. Few individuals flew before four days of age and thereafter flight profiles were similar for insects aged between four and 12 days. Mature females were more flight willing when kept as adults in mixed groups with males than when caged separately. There was no consistent effect on flight performance when insects were reared on rice varieties with different levels of leafhopper resistance. The flight activity of N. virescens was greater when leafhoppers were reared on mature, compared with young, rice plants. Leafhoppers reared through one generation on tungro-diseased rice plants were less willing to fly than individuals maintained on healthy plants of the same age and variety, whereas those tested after a 24-h access period to tungro-diseased plants were more flight-willing. The results are discussed in relation to the spread of tungro and to management interventions for the control of the disease.

ERK plays a regulatory role in induction of LTP by theta frequency stimulation and its modulation by beta-adrenergic receptors.

MAP kinase (ERK) translates cell surface signals into alterations in transcription. We have found that ERK also regulates hippocampal neuronal excitability during 5 Hz stimulation and thereby regulates forms of long-term potentiation (LTP) that do not require macromolecular synthesis. Moreover, ERK-mediated changes in excitability are selectively required for some forms of LTP but not others. ERK is required for the early phase of LTP elicited by brief 5 Hz stimulation, as well as for LTP elicited by more prolonged 5 Hz stimulation when paired with beta1-adrenergic receptor activation. By contrast, ERK plays no role in LTP elicited by a single 1 s 100 Hz train. Consistent with these results, we find that ERK is activated by beta-adrenergic receptors in CA1 pyramidal cell somas and dendrites.

Needle injection catheter delivery of the gene for an antibacterial agent inhibits neointimal formation.

Percutaneous transluminal coronary angioplasty is a routinely used non-surgical revascularization technique for patients with coronary artery disease. Up to 30% of patients undergoing coronary angioplasty develop a renarrowing of treated vessels, called restenosis. Smooth muscle cell proliferation is thought to be an important factor in restenosis; this leads to neointima formation and arterial lumen narrowing. Neointima may be reduced by the transfer of genes encoding proteins with antiproliferative effects. Cecropins are antimicrobial peptides with antiproliferative properties in mammalian cells. Cecropin A is one member of this family of peptides. In this article, a plasmid carrying the gene for the immature form, pre-pro-cecropin A, complexed with liposomes was locally delivered to perivascular tissue in a porcine arterial injury model using a needle injection catheter. Retention of the plasmid in the treated arteries was demonstrated at both 8 and 21 days following application. Transferred plasmid DNA was not detected in any other tissues analyzed. Pre-pro-cecropin A-specific transcripts could also be found in treated arteries. Balloon-injured vessels demonstrated significantly reduced neointima at 21 days in vessels treated with the pre-pro-cecropin A gene compared with neointimal area in those given a control gene (P < 0.05). The needle injection catheter appears to be useful for local intravascular gene delivery. In vivo gene transfer of cecropins may be of therapeutic relevance in restenosis prevention by limiting cell proliferation.

Rolipram, a type IV-specific phosphodiesterase inhibitor, facilitates the establishment of long-lasting long-term potentiation and improves memory.

In an attempt to improve behavioral memory, we devised a strategy to amplify the signal-to-noise ratio of the cAMP pathway, which plays a central role in hippocampal synaptic plasticity and behavioral memory. Multiple high-frequency trains of electrical stimulation induce long-lasting long-term potentiation, a form of synaptic strengthening in hippocampus that is greater in both magnitude and persistence than the short-lasting long-term potentiation generated by a single tetanic train. Studies using pharmacological inhibitors and genetic manipulations have shown that this difference in response depends on the activity of cAMP-dependent protein kinase A. Genetic studies have also indicated that protein kinase A and one of its target transcription factors, cAMP response element binding protein, are important in memory in vivo. These findings suggested that amplification of signals through the cAMP pathway might lower the threshold for generating long-lasting long-term potentiation and increase behavioral memory. We therefore examined the biochemical, physiological, and behavioral effects in mice of partial inhibition of a hippocampal cAMP phosphodiesterase. Concentrations of a type IV-specific phosphodiesterase inhibitor, rolipram, which had no significant effect on basal cAMP concentration, increased the cAMP response of hippocampal slices to stimulation with forskolin and induced persistent long-term potentiation in CA1 after a single tetanic train. In both young and aged mice, rolipram treatment before training increased long- but not short-term retention in freezing to context, a hippocampus-dependent memory task.