NDM-2 carbapenemase-producing Acinetobacter baumannii in the United Arab Emirates.

Screening 155 carbapenem non-susceptible Acinetobacter baumannii strains recovered in Abu Dhabi hospitals identified two metallo-ß-lactamase bla(NDM) gene-carrying isolates. They were isolated 4 months apart from the urine of a cancer patient previously treated in Egypt, Lebanon and in the United Arab Emirates. They were clonally related and carried the bla(NDM-2) gene recently identified in A. baumannii in Egypt and Israel. Sequences surrounding the bla(NDM-2) gene showed significant similarities with those associated with bla(NDM-1) in Enterobacteriaceae and A. baumannii. Repeated isolation of bla(NDM-2)-positive A. baumannii in the Middle East raises the possibility of the local emergence and spread of a unique clone.

Corticotropin-releasing hormone (CRH) expression and protein kinase A mediated CRH receptor signalling in an immortalized hypothalamic cell line.

Corticotropin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays an important role in the stress response in the hypothalamus. We describe the development of an immortalized hypothalamic cell line which expresses CRH. We hypothesized that this cell line would possess the relevant characteristics of parvocellular CRH-expressing neurones such as glucocorticoid receptor (GR) expression and vasopressin (VP) coexpression. For production of hypothalamic cells, embryonic day 19 rat pup hypothalami were dissected and dissociated into tissue culture dishes. They were immortalized by retrovirus-mediated transfer of the SV40 large T antigen gene at 3 days of culture and then screened for expression of CRH following dilution cloning. One cell line was chosen (IVB) which exhibited CRH-like immunoreactivity (CRH-LI) and expressed CRH, VP and CRH1 receptor RNA via the reverse transcriptase-polymerase chain reaction. In addition, the cell line expressed the neuronal marker, microtubule-associated protein-2. We verified that the CRH-LI from IVB cell lysates coeluted with CRH standard via reversed-phase high-performance liquid chromatography (HPLC). Furthermore, oxidation of the lysate converted its HPLC profile to that identical with oxidized CRH standard. In addition, IVB cells exhibited high affinity binding to CRH. Incubation of IVB cells with CRH lead to increases in cAMP levels and protein kinase A activity in a concentration-dependent manner. Incubation of IVB cells with CRH also resulted in increases in phospho-cyclic-AMP response element binding protein (CREB) immunostaining as detected by immunocytochemical analysis. Finally, CRH treatment of IVB cell lines has been linked to CREB-mediated gene expression as determined via the PathDetect CREB trans-reporting system. The characteristics of IVB cells, such as CRH and VP coexpression, GR expression and a biologically active CRH-R1-mediated signalling pathway, suggest that this neuronal cell line may serve as model of parvocellular CRH neurones.

Postnatal pre- and postexposure passive immunization strategies: protection of neonatal macaques against oral simian-human immunodeficiency virus challenge.

Simian-human immunodeficiency viruses (SHIV) allow the evaluation of antiviral strategies that target the envelope glycoproteins of the human immunodeficiency virus 1 (HIV-1) in macaques. We previously protected neonates from oral challenge with cell-free SHIV-vpu+ by passive immunization with synergistic human neutralizing monoclonal antibodies (mAbs) (Baba et al., Nat Med 6:200-206, 2000). mAbs were administered prenatally to pregnant dams and postnatally to the neonates. Here, we used solely postnatal or postexposure mAb treatment, thus significantly reducing the amount of mAbs necessary. All neonatal monkeys were also protected with these abbreviated mAb regimens. Our results are directly relevant for humans because we used mAbs that target HIV-1 envelope glycoproteins. Thus, the large-scale use of passive immunization with neutralizing mAbs may be feasible in human neonates. The mAbs, being natural human proteins, can be expected to have low toxicity. Passive immunization has promise to prevent intrapartum as well as milk-borne virus transmission from HIV-1-infected women to their infants.

DNA prime/protein boost vaccine strategy in neonatal macaques against simian human immunodeficiency virus.

Newborn macaques were vaccinated against a chimeric simian human immunodeficiency (SHIV) virus, SHIV-vpu+, by DNA priming and boosting with homologous HIV-1 gp160. Following SHIV-vpu+ challenge, containment of infection was observed in 4 of 15 animals given DNA priming/protein boost vaccination and in three of four animals given gp160 boosts only. Rechallenge with homologous virus of six animals that contained the first challenge virus resulted in rapid viral clearance or low viral loads. Upon additional rechallenge with heterologous, pathogenic SHIV89.6P, four of these six animals maintained normal CD4+ T-cell counts with no or limited SHIV89.6P infection. Our data suggest that humoral and cellular immune mechanisms may have contributed to the containment of SHIV89.6P; however, viral interference with SHIV-vpu+ could also have played a role. Our results indicate that immunogenicity and efficacy of candidate AIDS vaccines are not affected when vaccination is initiated during infancy as compared with later in life.

Protection of neonatal macaques against experimental SHIV infection by human neutralizing monoclonal antibodies.

Neonatal macaques were completely protected against oral challenge with SHIV-vpu+, a simian-human immunodeficiency virus that encodes the envelope gene of a laboratory-adapted HIV strain, by pre- and post-natal treatment with a triple combination of human neutralizing monoclonal antibodies (mAbs). The mAbs were directed either against the CD4 binding site, a glycosylation-dependent gp120 epitope, or against a linear epitope on gp41. This triple combination was highly synergistic in vitro and neutralized primary HIV completely. Subsequently, oral challenge was performed with pathogenic SHIV89.6P, an animal-passaged variant of a chimeric virus that encodes the envelope gene of the primary, dual-tropic HIV89.6. Only post-natal treatment with a similar triple mAb combination was used. One out of 4 mAb-treated infants was completely protected from infection. In the other 3 treated animals, there was a tendency towards lower peak viral RNA loads compared with untreated controls. Two out of 4 mAb-treated infants maintained normal CD4+ T-cell numbers, in contrast to all controls that had steep declines at 2 weeks post-challenge. We conclude that the triple mAb combination significantly protected the neonates, even against mucosal challenge with pathogenic SHIV89.6P. Passively administered synergistic human mAbs may play a role in preventing mother-infant transmission of HIV, both against intrapartum transmission as well as against infection through breast milk. As passive immunization is a tool to assess correlates of immune protection, we conclude that the epitopes recognized by the mAbs in our combinations are important for AIDS vaccine development. Future passive immunization studies may reveal other important conserved epitopes.

Passive immunization against oral AIDS virus transmission: an approach to prevent mother-to-infant HIV-1 transmission?

To develop immunoprophylaxis regimens against mother-to-child human immunodeficiency virus type 1 (HIV-1) transmission, we established a simian-human immunodeficiency virus (SHIV) model in neonatal macaques that mimics intrapartum mucosal virus exposure (T.W. Baba, J. Koch, E.S. Mittler et al: AIDS Res Hum Retroviruses 10:351-357, 1994). We protected four neonates from oral SHIV-vpu+ challenge by ante- and postpartum treatment with a synergistic triple combination of immunoglobulin (Ig) G1 human anti-HIV-1 neutralizing monoclonal antibodies (mAbs) (T.W. Baba, V. Liska, R. Hofmann-Lehmann et al: Nature Med 6:200-206, 2000), which recognize the CD4-binding site of Env, a glycosylation-dependent gp120, or a linear gp41 epitope. Two neonates that received only postpartum mAbs were also protected from oral SHIV-vpu+ challenge, indicating that postpartum treatment alone is sufficient. Next, we evaluated a similar mAb combination against SHIV89.6P, which encodes env of primary HIV89.6. One of four mAb-treated neonates was protected from infection and two maintained normal CD4+ T-cell counts. We conclude that the epitopes recognized by the three mAbs are important determinants for achieving protection. Combination immunoprophylaxis with synergistic mAbs seems promising to prevent maternal HIV-1 transmission in humans.

Postnatal passive immunization of neonatal macaques with a triple combination of human monoclonal antibodies against oral simian-human immunodeficiency virus challenge.

To develop prophylaxis against mother-to-child human immunodeficiency virus (HIV) transmission, we established a simian-human immunodeficiency virus (SHIV) infection model in neonatal macaques that mimics intrapartum mucosal virus exposure (T. W. Baba et al., AIDS Res. Hum. Retroviruses 10:351-357, 1994). Using this model, neonates were protected from mucosal SHIV-vpu(+) challenge by pre- and postnatal treatment with a combination of three human neutralizing monoclonal antibodies (MAbs), F105, 2G12, and 2F5 (Baba et al., Nat. Med. 6:200-206, 2000). In the present study, we used this MAb combination only postnatally, thereby significantly reducing the quantity of antibodies necessary and rendering their potential use in humans more practical. We protected two neonates with this regimen against oral SHIV-vpu(+) challenge, while four untreated control animals became persistently infected. Thus, synergistic MAbs protect when used as immunoprophylaxis without the prenatal dose. We then determined in vitro the optimal MAb combination against the more pathogenic SHIV89.6P, a chimeric virus encoding env of the primary HIV89.6. Remarkably, the most potent combination included IgG1b12, which alone does not neutralize SHIV89.6P. We administered the combination of MAbs IgG1b12, 2F5, and 2G12 postnatally to four neonates. One of the four infants remained uninfected after oral challenge with SHIV89.6P, and two infants had no or a delayed CD4(+) T-cell decline. In contrast, all control animals had dramatic drops in their CD4(+) T cells by 2 weeks postexposure. We conclude that our triple MAb combination partially protected against mucosal challenge with the highly pathogenic SHIV89.6P. Thus, combination immunoprophylaxis with passively administered synergistic human MAbs may play a role in the clinical prevention of mother-to-infant transmission of HIV type 1.

Primate and feline lentivirus vector RNA packaging and propagation by heterologous lentivirus virions.

Development of safe and effective gene transfer systems is critical to the success of gene therapy protocols for human diseases. Currently, several primate lentivirus-based gene transfer systems, such as those based on human and simian immunodeficiency viruses (HIV/SIV), are being tested; however, their use in humans raises safety concerns, such as the generation of replication-competent viruses through recombination with related endogenous retroviruses or retrovirus-like elements. Due to the greater phylogenetic distance from primate lentiviruses, feline immunodeficiency virus (FIV) is becoming the lentivirus of choice for human gene transfer systems. However, the safety of FIV-based vector systems has not been tested experimentally. Since lentiviruses such as HIV-1 and SIV have been shown to cross-package their RNA genomes, we tested the ability of FIV RNA to get cross-packaged into primate lentivirus particles such as HIV-1 and SIV, as well as a nonlentiviral retrovirus such as Mason-Pfizer monkey virus (MPMV), and vice versa. Our results reveal that FIV RNA can be cross-packaged by primate lentivirus particles such as HIV-1 and SIV and vice versa; however, a nonlentivirus particle such as MPMV is unable to package FIV RNA. Interestingly, FIV particles can package MPMV RNA but cannot propagate the vector RNA further for other steps of the retrovirus life cycle. These findings reveal that diverse retroviruses are functionally more similar than originally thought and suggest that upon coinfection of the same host, cross- or copackaging may allow distinct retroviruses to generate chimeric variants with unknown pathogenic potential.

Virion-associated HIV-1 Vpr: variable amount in virus particles derived from cells upon virus infection or proviral DNA transfection.

Human immunodeficiency virus type-1 (HIV-1) Vpr is a virion-associated protein implicated to have a role in AIDS pathogenesis. In regard to the amount of Vpr incorporated into virus particles, the published data vary widely. To address this, we quantitated Vpr in virus particles derived from diverse sources that are used to evaluate the biological effect of Vpr. Virus particles from infected cells showed only a small amount of Vpr. Interestingly, virus particles from cells cotransfected with HIV-1 proviral DNA lacking Vpr coding sequences (NLDeltaVpr) and a Vpr expression plasmid showed a drastic increase (29.4-fold) in the incorporation of Vpr. Furthermore, cotransfection involving NLDeltaVpr and different concentrations of Vpr expression plasmid resulted in virus particles containing Vpr in proportion to the Vpr expression plasmid used. The differences in virus particles with respect to Vpr as revealed by these studies should be taken into account in assessing the effect of Vpr.

Enhancement of mucosal immune response against HIV-1 Gag by DNA immunization.

In order to examine the feasibility of Gag-expression DNA as a potential candidate for HIV vaccine using a mouse model, we injected DNA into mice either intramuscularly or by using a gene gun. Both methods induced a low level of antibody production. However, after booster immunization with p24 protein emulsified with complete Freund's adjuvant via a footpad, we found that only the preceding intramuscular DNA immunization induced an anti-Gag Th1-type (IgG(2a)) antibody response, in addition to the enhancement of a Th2-type (IgG(1)) antibody response. Importantly, when mice were boosted intranasally with p24 and cholera toxin, intramuscular DNA injection was found to enhance both systemic and mucosal Gag-specific immune responses. These results indicate that intramuscular DNA immunization confers the inducibility of memory cells, which circulate around various mucosal tissues. Therefore, intramuscular DNA priming, followed by a mucosal booster immunization, could be considered as a regimen applicable to HIV vaccine.

Intravirion display of a peptide corresponding to the dimer structure of protease attenuates HIV-1 replication.

Current treatment of HIV-1-infected individuals involves the administration of several drugs, all of which target either the reverse transcriptase or the protease activity of the virus. Unfortunately, the benefits of such treatments are compromised by the emergence of viruses exhibiting resistance to the drugs. This situation warrants new approaches for interfering with virus replication. Considering the activation of protease in the virus particles, a novel strategy to inhibit HIV-1 replication was tested targeting the dimerization domain of the protease. To test this idea, we have selected four residues from the C terminus of HIV-1 protease that map to the dimer interface region of the enzyme. We have exploited Vpr to display the peptides in the virus particles. The chimeric Vpr exhibited expression and virion incorporation similar to wildtype Vpr. The virus derived from the HIV-1 proviral DNA containing chimeric Vpr sequences registered a reduced level of replication in CEM and CEM X 174 cells in comparison with viruses containing wildtype Vpr. Similar results were observed in a single-round replication assay. These results suggest that the intravirion display of peptides targeting viral proteins is a powerful approach for developing antiviral agents and for dissecting the dynamic interactions between structural proteins during virus assembly and disassembly.

CD44 enhances neuregulin signaling by Schwann cells.

We describe a key role for the CD44 transmembrane glycoprotein in Schwann cell-neuron interactions. CD44 proteins have been implicated in cell adhesion and in the presentation of growth factors to high affinity receptors. We observed high CD44 expression in early rat neonatal nerves at times when Schwann cells proliferate but low expression in adult nerves, where CD44 was found in some nonmyelinating Schwann cells and to varying extents in some myelinating fibers. CD44 constitutively associated with erbB2 and erbB3, receptor tyrosine kinases that heterodimerize and signal in Schwann cells in response to neuregulins. Moreover, CD44 significantly enhanced neuregulin-induced erbB2 phosphorylation and erbB2-erbB3 heterodimerization. Reduction of CD44 expression in vitro resulted in loss of Schwann cell-neurite adhesion and Schwann cell apoptosis. CD44 is therefore crucial for maintaining neuron-Schwann cell interactions at least partly by facilitating neuregulin-induced erbB2-erbB3 activation.

Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection.

Although maternal human immunodeficiency virus type 1 (HIV-1) transmission occurs during gestation, intrapartum and postpartum (by breast-feeding), 50-70% of all infected children seem to acquire HIV-1 shortly before or during delivery. Epidemiological evidence indicates that mucosal exposure is an important aspect of intrapartum HIV transmission. A simian immunodeficiency virus (SIV) macaque model has been developed that mimics the mucosal exposure that can occur during intrapartum HIV-1 transmission. To develop immunoprophylaxis against intrapartum HIV-1 transmission, we used SHIV-vpu+ (refs. 5,6), a chimeric simian-human virus that encodes the env gene of HIV-IIIB. Several combinations of human monoclonal antibodies against HIV-1 have been identified that neutralize SHIV-vpu+ completely in vitro through synergistic interaction. Here, we treated four pregnant macaques with a triple combination of the human IgG1 monoclonal antibodies F105, 2G12 and 2F5. All four macaques were protected against intravenous SHIV-vpu+ challenge after delivery. The infants received monoclonal antibodies after birth and were challenged orally with SHIV-vpu+ shortly thereafter. We found no evidence of infection in any infant during 6 months of follow-up. This demonstrates that IgG1 monoclonal antibodies protect against mucosal lentivirus challenge in neonates. We conclude that epitopes recognized by the three monoclonal antibodies are important determinants for achieving substantial protection, thus providing a rational basis for AIDS vaccine development.

The organization of preoptic-medullary circuits in the male rat: evidence for interconnectivity of neural structures involved in reproductive behavior, antinociception and cardiovascular regulation.

The present studies used anatomical tract-tracing techniques to delineate the organization of pathways linking the medial preoptic area and the ventral medulla, two key regions involved in neuroendocrine, autonomic and sensory regulation. Wheatgerm agglutinin-horseradish peroxidase injections into the ventromedial medulla retrogradely labeled a large number of neurons in the medial preoptic area, including both the median and medial preoptic nuclei. The termination pattern of preoptic projections to the medulla was mapped using the anterograde tracers Phaseolus vulgaris leucoagglutinin and biotinylated dextran amine. Tracer injections into the preoptic area produced a dense plexus of labeled fibers and terminals in the ventromedial and ventrolateral pons and medulla. Within the caudal pons/rostral medulla, medial preoptic projections terminated heavily in the nucleus raphe magnus; strong anterograde labeling was also present in the pontine reticular field. At mid-medullary levels, labeled fibers focally targeted the nucleus paragigantocellularis, in addition to the heavy fiber labeling present in the midline raphe nuclei. By contrast, very little labeling was observed in the caudal third of the medulla. Experiments were also conducted to map the distribution of ventral pontine and medullary neurons that project to the medial preoptic area. Wheatgerm agglutinin-horseradish peroxidase injections in the preoptic area retrogradely labeled a significant population of neurons in the ventromedial and ventrolateral medulla. Ascending projections from the medulla to the preoptic area were organized along rostral-caudal, medial-lateral gradients. In the caudal pons/rostral medulla, retrogradely labeled cells were aggregated along the midline raphe nuclei; no retrograde labeling was present laterally at this level. By contrast, in the caudal half of the medulla, cells retrogradely labeled from the medial preoptic area were concentrated as a discrete zone dorsal to the lateral reticular nucleus; labeled cells were not present in the ventromedial medulla at this level. The present findings suggest that the medial preoptic area and ventral midline raphe nuclei share reciprocal connections that are organized in a highly symmetrical fashion. By contrast, preoptic-lateral medullary pathways are not reciprocal. These preoptic-brainstem circuits may participate in antinociceptive, autonomic and reproductive behaviors.

Neutralizing antibody-independent containment of immunodeficiency virus challenges by DNA priming and recombinant pox virus booster immunizations.

Eight different protocols were compared for their ability to raise protection against immunodeficiency virus challenges in rhesus macaques. The most promising containment of challenge infections was achieved by intradermal DNA priming followed by recombinant fowl pox virus booster immunizations. This containment did not require neutralizing antibody and was active for a series of challenges ending with a highly virulent virus with a primary isolate envelope heterologous to the immunizing strain.

Oral transmission of primate lentiviruses.

Oral transmission of human immunodeficiency virus type 1 (HIV-1) is well documented in children who become infected postnatally through breast milk. In contrast, epidemiologic surveys have yielded conflicting data regarding oral HIV-1 transmission among adults, even though case reports have described seroconversion and the development of AIDS in adults whose only risk was oral-genital contact. To study oral virus transmission in primate models, we exposed rhesus macaques of various ages to cell-free simian immunodeficiency virus (SIV), including uncloned and molecularly cloned viruses. In neonates, viremia and AIDS developed after nontraumatic oral exposure to several SIV strains. Furthermore, chimeric simian human immunodeficiency viruses containing the HIV-1 envelope can also cross intact upper gastrointestinal mucosal surfaces in neonates. In adult macaques, infection and AIDS have resulted from well-controlled, nontraumatic, experimental oral exposure to different strains of SIV. These findings have implications for the risks of HIV-1 transmission during oral-genital contact.

HIV-1 Gag shares a signature motif with annexin (Anx7), which is required for virus replication.

Genetic and biochemical analyses of the Gag protein of HIV-1 indicate a crucial role for this protein in several functions related to viral replication, including viral assembly. It has been suggested that Gag may fulfill some of the functions by recruiting host cellular protein(s). In our effort to identify structural and functional homologies between Gag and cellular cytoskeletal and secretory proteins involved in transport, we observed that HIV-1 Gag contains a unique PGQM motif in the capsid region. This motif was initially noted in the regulatory domain of synexin the membrane fusion protein of Xenopus laevis. To evaluate the functional significance of the highly conserved PGQM motif, we introduced alanine (A) in place of individual residues of the PGQM and deleted the motif altogether in a Gag expression plasmid and in an HIV-1 proviral DNA. The proviral DNA containing mutations in the PGQM motif showed altered expression, assembly, and release of viral particles in comparison to parental (NL4-3) DNA. When tested in multiple- and single-round replication assays, the mutant viruses exhibited distinct replication phenotypes; the viruses containing the A for the G and Q residues failed to replicate, whereas A in place of the P and M residues did not inhibit viral replication. Deletion of the tetrapeptide also resulted in the inhibition of replication. These results suggest that the PGQM motif may play an important role in the infection process of HIV-1 by facilitating protein-protein interactions between viral and/or viral and cellular proteins.

Region-specific astrogliosis in brains of mice heterozygous for mutations in the neurofibromatosis type 1 (Nf1) tumor suppressor.

Brains from human neurofibromatosis type 1 (NF1) patients show increased expression of glial fibrillary acidic protein (GFAP), consistent with activation of astrocytes (M.L. Nordlund, T.A. Rizvi, C.I. Brannan, N. Ratner, Neurofibromin expression and astrogliosis in neurofibromatosis (type 1) brains, J. Neuropathol. Exp. Neurology 54 (1995) 588-600). We analyzed brains from transgenic mice in which the Nf1 gene was targeted by homologous recombination. We show here that, in all heterozygous mice analyzed, there are increased numbers of astrocytes expressing high levels of GFAP in medial regions of the periaqueductal gray and in the nucleus accumbens. More subtle, but significant, changes in the number of GFAP positive astrocytes were observed in the hippocampus in 60% of mutant mice analyzed. Astrocytes with elevated GFAP were present at 1 month, 2 months, 6 months and 12 months after birth. Most brain regions, including the cerebellum, basal ganglia, cerebral cortex, hypothalamus, thalamus, cortical amygdaloid area, and white matter tracts did not show any gliotic changes. No evidence of degenerating neurons was found using de Olmos' cupric silver stain. We conclude that Nf1/nf1 mice provide a model to study astrogliosis associated with neurofibromatosis type 1.

Ruffling membrane, stress fiber, cell spreading and proliferation abnormalities in human Schwannoma cells.

Schwannomas are peripheral nerve tumors that typically have mutations in the NF2 tumor suppressor gene. We compared cultured schwannoma cells with Schwann cells from normal human peripheral nerves (NHSC). Both cell types expressed specific antigenic markers, interacted with neurons, and proliferated in response to glial growth factor, confirming their identity as Schwann cells. Schwannoma cells frequently had elevated basal proliferation compared to NHSC. Schwannoma cells also showed spread areas 5-7-fold greater than NHSC, aberrant membrane ruffling and numerous, frequently disorganized stress fibers. Dominant negative Rac inhibited schwannoma cell ruffling but had no apparent effect on NHSC. Schwannoma cell stress fibers were inhibited by C3 transferase, tyrphostin A25, or dominant negative RhoA. These data suggest that the Rho and Rac pathways are abnormally activated in schwannoma cells. Levels of ezrin and moesin, proteins related to the NF2 gene product, merlin, were unchanged in schwannoma cells compared to NHSC. Our findings demonstrate for the first time that cell proliferation and actin organization are aberrant in schwannoma cells. Because NF2 is mutant in most or all human schwannomas, we postulate that loss of NF2 contributes to the cell growth and cytoskeletal dysfunction reported here.

Fos expression in rat pontine tegmental neurons following activation of the medial preoptic area.

Fos immunohistochemistry was used to map the distribution of pontine neurons excited by activation of the medial preoptic area (MPO). Although we have previously shown that Barrington's nucleus receives a very dense focal input from the MPO, electrical stimulation of the preoptic area unexpectedly induced very little Fos expression in Barrington's neurons. These results suggest that the MPO-->Barrington's projection utilizes a transmitter(s) that does not involve transduction of the Fos protein; alternatively, MPO afferents to Barrington's nucleus may be inhibitory in nature. As Barrington's nucleus plays a critical role in micturition, MPO projections to Barrington's nucleus may regulate voiding reflexes during sexual behavior. Interestingly, while the locus coeruleus (LC) proper receives only a sparse projection from the MPO, extensive Fos expression was present in LC. The finding of Fos immunoreactive LC neurons suggests that the excitatory influence of MPO may regulate LC neuronal activity and NE release during reproductive behaviors.