Efficacy of hydrogen peroxide wipes for decontamination of AZD1222 adenovirus COVID-19 vaccine strain on pharmaceutical industry materials.

This study aimed to evaluate the performance of accelerated hydrogen peroxide® wipes (HPW) for decontamination of the chimpanzee adenovirus AZD1222 vaccine strain used in the production of recombinant COVID-19 vaccine in a pharmaceutical industry. Two matrices were tested on stainless-steel (SS) and low-density-polyethylene (LDP) surfaces: formulated recombinant COVID-19 vaccine (FCV) and active pharmaceutical ingredient (API). The samples were spiked, dried and the initial inoculum, possible residue effect (RE) and titre reduction after disinfection with HPW were determined. No RE was observed. The disinfection procedure with HPW resulted in complete decontamination the of AZD1222 adenovirus strain in FCV (≥7·46 and ≥7·49 log10 infectious unit [IFU] ml-1 for SS and LDP carriers respectively) and API (≥8·79 and ≥8·78 log10 IFU ml-1 for SS and LDP carriers respectively). In conclusion, virucidal activity of HPW was satisfactory against the AZD1222 adenovirus strain and can be a good option for disinfection processes of SS and LPD surfaces in pharmaceutical industry facilities during recombinant COVID-19 vaccine production. This procedure is simple and can be also applied on safety unit cabins and sampling bags made of LDP as well.

Case report of Curtobacterium isolated from a catheter tip sample misidentified as Cronobacter.

The Curtobacterium genus is a member of the family Microbacteriaceae, and Curtobacterium species are recognized as plant pathogens. The aim of this study was to investigate a dubious result of species identification for an infection located on a catheter tip of a patient with Covid-19. A strain isolated from a catheter tip sample, identified by VITEK® 2 as Cronobacter spp., was submitted to polyphasic analysis: Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) using VITEK® MS, real-time polymerase chain reaction targeting dnaG gene, and 16S rRNA full gene Sanger sequencing analysis for confirmation. The strain presented negative result using qPCR and could not identified by MALDI-TOF MS. 16S rRNA full gene Sanger sequencing analysis identified the strain as Curtobacterium spp. The Gram-variable characteristic (Gram-negative instead of Gram-positive) of the isolated strain was the responsible for the misidentification by VITEK® 2 and VITEK® MS did not identify the strain. 16S rRNA full gene sequencing analysis identified the strain as Curtobacterium genus, but other complementary techniques are necessary to identify at species level.

Evaluation of hydrogen peroxide efficacy against AZD1222 chimpanzee adenovirus strain in the recombinant COVID-19 vaccine for application in cleaning validation in a pharmaceutical manufacturing industry.

This study aimed to evaluate the performance of hydrogen peroxide vapour (HPV) to inactivate the chimpanzee adenovirus AZD1222 vaccine strain used in the production of recombinant COVID-19 vaccine for application in cleaning validation in pharmaceutical industries production areas. Two matrixes were tested: formulated recombinant COVID-19 vaccine (FCV) and active pharmaceutical ingredient (API). The samples were dried on stainless steel and exposed to HPV in an isolator. One biological indicator with population >106 Geobacillus stearothermophilus spores was used to validate the HPV decontamination cycle as standard. HPV exposure resulted in complete virus inactivation in FVC (≥5·03 log10 ) and API (≥6·40 log10 ), showing HPV efficacy for reducing chimpanzee adenovirus AZD1222 vaccine strain. However, the optimum concentration and contact time will vary depending on the type of application. Future decontamination studies scaling up the process to the recombinant COVID-19 vaccine manufacturing areas are necessary to evaluate if the HPV will have the same or better virucidal effectivity in each specific production area. In conclusion, HPV showed efficacy for reducing AZD1222 chimpanzee adenovirus strain and can be a good choice for pharmaceutical industries facilities disinfection during recombinant COVID-19 vaccine production.

Cytotoxicity profile of Cronobacter species isolated from food and clinical specimens in Brazil.

AIMS: The objective of this study was to evaluate the cytotoxic activity of Cronobacter strains isolated from foods (n = 50) and clinical samples (n = 6) in Brazil and genotype selected strains (n = 18) using multi-locus sequence typing (MLST) METHODS AND RESULTS: The cytotoxic activity of C. sakazakii (n = 29), C. dublinensis (n = 13), C. malonaticus (n = 6), C. turicensis (n = 6) and C. muytjensii (n = 2) was screened using Vero, RK13, Hep2c, NCTC clone 929 and BHK-21 cell lines. Selected Cronobacter strains were assigned to C. sakazakii ST 21, C. turicensis ST 252, C. sakazakii ST 647, and three newly assigned STs: C. turicensis STs 738-740. The maximum death caused by non-heat-treated filtrates was 20·4, 86·2, 47·0 and 84·0%, in Vero, RK13, Hep2c and NCTC clone 929 cells, respectively. These were caused by C. sakazakii strains C291 and C292 (ST 494) which had been isolated during neonatal Cronobacter meningitis infection, and C110 (ST 395) isolated from flaxseed flour. Thermal treatment (100°C/20 min) significantly reduced the cytotoxicity activity in NCTC clone 929 and Vero cells (P ≤ 2 × 10-6 ), but not in RK13 (P = 0·12) and Hep2c (P = 0·85), indicating the cytotoxin(s) were probably proteinaceous. Electron microscopy revealed that cytotoxic compounds from C. sakazakii induced several cell death characteristics, including loss of cell-cell contact, microvilli reduction and cellular lysis. Autophagic vacuoles and mitochondrial damage were the most common ultrastructural features observed. CONCLUSIONS: It was concluded that Cronobacter strains, especially C. sakazakii, could produce heat-labile cytotoxic compounds in cell filtrates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study providing insights into the pathogenesis of the Cronobacter genus. Cytotoxins were identified in excreted filtrates of C. sakazakii strains isolated from food and clinical specimens. The presence of Cronobacter strains that can produce cytotoxins in foods can be a potential threat to human health and highlight the need for high levels of hygiene.

Genetic diversity of Listeria monocytogenes serotype 1/2a strains collected in Brazil by Multi-Virulence-Locus Sequence Typing.

Listeria monocytogenes is an opportunistic pathogen with the ability to adapt to different environmental conditions, resulting in safety issues for food producers. Foods contaminated by L. monocytogenes can represent a risk if consumed by susceptible individuals such as elderly, pregnant women and the immunocompromised. The aim of this study was to evaluate the genetic diversity of a collection of L. monocytogenes isolated from different matrices in Brazil during the period of 1979-2015. A total of 51 L. monocytogenes serotype 1/2a strains isolated from clinical samples (n = 3) and food samples (n = 48) were characterized by Multi-Virulence-Locus Sequence Typing (MVLST). The strains were assigned to nine virulence types (VT): VT-11 (n = 3, 5·9%), VT-45 (n = 27, 52·9%), VT-59 (n = 11, 21·6%), VT-68 (n = 3, 5·9%), VT-94 (n = 2, 3·9%), VT-107 (n = 2, 3·9%), VT-184 (n = 1, 1·9%), VT-185 (n = 1, 1·9%) and VT-186 (n = 1, 1·9%); and four of them (VT-11, VT-45, VT-59 and VT-68) have already been associated with cases of listeriosis worldwide. The VT-11, VT-59 (Epidemic Clone V) and VT-186 were identified in blood culture samples, as well as in different classes of foods. It is recommended that the epidemiological surveillance agencies evaluate the risk that foods contaminated with L. monocytogenes VTs pose to susceptible populations.

Microbiota oral e retal de calitriquídeos (Callithrix sp) em área antropizada de Mata Atlântica, Rio de Janeiro, Brasil / [Oral and rectal microbiota of callitrichids (Callithrix sp) in an anthropized area of the Atlantic Forest in Rio de Janeiro, Brazil]

A proximidade dos primatas não humanos (PNH) com o ser humano pode ser considerada um fator de risco para transmissão de bactérias entre essas duas populações. Neste estudo, foi investigada a microbiota anfibiôntica aeróbica oral e retal de calitriquídeos em um fragmento de Mata Atlântica localizado no Rio de Janeiro, Brasil, e foram realizados testes fenotípicos para detecção de bactérias multirresistentes nos isolados encontrados. Foram capturados 14 calitriquídeos e coletadas 21 amostras (14 de cavidade oral e sete de cavidade retal) em dois pontos da mata próximos às habitações humanas. As espécies mais frequentes, na cavidade oral, foram Klebsiella oxytoca (50,0%), K. pneumoniae (28,6%), Kluyvera ascorbata (21,4%) e Stenotrophomonas maltophilia (21,4%) e, na cavidade retal, K. pneumoniae (85,7%), Escherichia coli (28,6%) e Enterobacter spp. (42,9%). Todos os 48 isolados da família Enterobacteriaceae foram negativos para ESBL (betalactamase de espectro ampliado), mostrando-se não produtores da enzima nos dois métodos utilizados: disco-aproximação e método de detecção automatizado. Na pesquisa de ERC (enterobactérias resistentes a carbapenêmicos), esses mesmos isolados não apresentaram resistência aos antibióticos imipenem, meropenem e ertapenem. Todas as bactérias isoladas apresentam um potencial zoonótico, o que representa um risco à saúde pública e à conservação das espécies.(AU)

Proximity of nonhuman primates (NHP) to humans can be considered a risk factor for transmission of pathogens between these two populations. This study investigated the oral and rectal aerobic bacterial microbiota of marmosets in an anthropized area of the Atlantic Forest located in Rio de Janeiro, Brazil, and performed phenotypic tests for detection of multidrug-resistant bacteria. Twenty-one samples (14 from the oral cavity and seven from the rectum) were collected from 14 Callithrix sp. captured in two sites of the forest near human dwellings. The most frequent species identified from the oral cavity swabs were Klebsiella oxytoca (50.0%), K. pneumoniae (28.6%), Kluyvera ascorbata (21.4%) and Stenotrophomonas maltophilia (21.4%), whereas the species most commonly identified from the rectum swabs were K. pneumoniae (85.7%), Enterobacter spp. (42.9%) and Escherichia coli (28.6%). All isolates of family Enterobacteriaceae showed no extended spectrum ß-lactamase production by disk-diffusion and automated detection tests. In the search for carbapenem-resistant enterobacteriaceae these isolates presented no resistance to the imipenem, meropenem and ertapenem antibiotics. The isolate of Staphylococcus aureus was susceptible to oxacillin and the isolate of Enterococcus was susceptible to vancomycin. All isolated bacteria showed zoonotic potential, thus posing a risk to species conservation and public health.(AU)

Occurrence of total coliforms, Escherichia coli and Cronobacter species in commercially available 20 l bottled drinking water sold in Rio de Janeiro State, Brazil.

Cronobacter infections of infants are commonly regarded as due to the ingestion of contaminated feed. The aim of this study was to determine the occurrence of Cronobacter, total coliforms and Escherichia coli in different brands of natural mineral waters as sold in 20 l returnable bottles in Rio de Janeiro, Brazil. The quantification of total coliforms and E. coli was performed by Most Probable Number. The detection of Cronobacter was as according to the ISO 22964:2017 and Bacteriological Analytical Manual/FDA. Molecular characterization of Cronobacter isolates was performed by real-time PCR and by multi-locus sequence typing. The antibiotic susceptibility profile was determined and biofilm production was evaluated in polystyrene microplates. Total coliforms and E. coli were detected in 13 (39·4%) and 2 (6·1%) of the 33 lots analysed respectively, and were considered unsatisfactory for human consumption according to Brazilian law. One (3·0%) lot showed contamination by C. malonaticus ST440 (Cronobacter MLST Databases accession no. ID 2646). The strain was susceptible to all (n = 13) antibiotics tested and only formed a weak biofilm. Since there is a high consumption of natural mineral waters by elderly and immunosuppressed persons, epidemiological surveillance agencies should be aware of the risk that these waters may represent for these groups. SIGNIFICANCE AND IMPACT OF THE STUDY: Cronobacter malonaticus ST440 was isolated from 20 l bottled drinking natural mineral waters sold in markets in Rio de Janeiro State, Brazil, and can be a potential threat to human health, particularly for neonates. Thirteen lots (39·4%) were unsatisfactory for human consumption due to the presence of total coliforms and/or Escherichia coli.

Role of the dorsal periaqueductal gray in posttraumatic stress disorder: mediation by dopamine and neurokinin.

In susceptible individuals, exposure to intensely traumatic life events can lead to the development of posttraumatic stress disorder (PTSD), including long-term dysregulation of the contextual processing of aversive stimuli, the overgeneralization of learned fear, and impairments in the ability to learn or respond to safety signals. The neuropathophysiological changes that underlie PTSD remain incompletely understood. Attention has focused on forebrain structures associated with fear processing. Here we consider evidence from human and animal studies that long-lasting changes in functional connectivity between the midbrain periaqueductal gray (dPAG) and amygdala may be one of the precipitating events that contribute to PTSD. Long-lasting neuroplastic changes in the dPAG can persist after a single aversive stimulation and are pharmacologically labile. The early stage (at least up to 24 h post-stimulation) involves neurokinin-1 receptor-mediated events in the PAG and amygdala and is also regulated by dopamine, both of which are mainly involved in transferring ascending aversive information from the dPAG to higher brain structures, mainly the amygdala. Changes in the functional connectivity within the dPAG-amygdala circuit have been reported in PTSD patients. We suggest that further investigations of plasticity and pharmacology of the PAG-amygdala network provide a promising target for understanding pathophysiological circuitry that underlies PTSD in humans and that dopaminergic and neurokininergic drugs may have a potential for the treatment of psychiatric disorders that are associated with a dysfunctional dPAG.

Assessment of viral and bacterial contamination of fresh and ripened semi-hard cheeses.

This study aimed to evaluate viral and bacterial contamination from typical Brazilian cheeses, such as Minas (fresh) and Prato (ripened), commercially obtained in the Greater Metropolitan Region of the State of Rio de Janeiro, Brazil. Minas [30], Prato [30] and sliced Prato [30] cheese samples were investigated for norovirus genogroup I and II (NoV GI-II) and human adenovirus (HAdV) by direct nucleic acid extraction using TRIzol and amplification by TaqMan based quantitative polymerase chain reaction. Listeria monocytogenes, Salmonella spp., coagulase-positive staphylococci (CPS) and fecal coliforms were also assessed by using standard counting methods. NoV GI and GII were detected in one sample (1.1%) each and HAdV in nine samples (10.0%) while bacteriological analysis revealed five samples (5.5%) contaminated with L. monocytogenes, 27 (30.0%) with fecal coliforms and 10 (11.1%) with CPS. Salmonella spp. was not detected in any sample. Viruses were detected in 11 samples (12.2%), of which 9 met the microbiological criteria used to evaluate the microbiological quality of the cheeses, stressing the importance of considering virological parameters for monitoring this food matrix.

Behavioral sensitization induced by dorsal periaqueductal gray electrical stimulation is counteracted by NK1 receptor antagonism in the ventral hippocampus and central nucleus of the amygdala.

A single threatening experience may change the behavior of an animal in a long-lasting way and elicit generalized behavioral responses to a novel threatening situation that is unrelated to the original aversive experience. Electrical stimulation (ES) of the dorsal periaqueductal gray (dPAG) produces a range of defensive reactions, characterized by freezing, escape, and post-stimulation freezing (PSF). The latter reflects the processing of ascending aversive information to prosencephalic structures, including the central nucleus of the amygdala (CeA), which allows the animal to evaluate the consequences of the aversive situation. This process is modulated by substance P (SP) and its preferred receptor, neurokinin 1 (NK1). The ventral hippocampus (VH) has been associated with the processing of aversive information and expression of emotional reactions with negative valence, but the participation of the VH in the expression of these defensive responses has not been investigated. The VH is rich in NK1 receptor expression and has a high density of SP-containing fibers. The present study examined the role of NK1 receptors in the VH in the expression of defensive responses and behavioral sensitization that were induced by dPAG-ES. Rats were implanted with an electrode in the dPAG for ES, and a cannula was implanted in the VH or CeA for injections of vehicle (phosphate-buffered saline) or the NK1 receptor antagonist spantide (100 pmol/0.2 µL. Spantide reduced the duration of PSF that was evoked by dPAG-ES, without changing the aversive freezing or escape thresholds. One and 7 days later, exploratory behavior was evaluated in independent groups of rats in the elevated plus maze (EPM). dPAG-ES in rats that received vehicle caused higher aversion to the open arms of the EPM compared with rats that did not receive dPAG stimulation at both time intervals. Injections of spantide in the VH or CeA prevented the proaversive effects of dPAG-ES in the EPM only 1 day later. These findings suggest that NK1 receptors are activated in both the VH and CeA during the processing of aversive information that derives from dPAG-ES. As previously shown for the CeA, SP/NK1 receptors in the VH are recruited during PSF that is evoked by dPAG-ES, suggesting that a 24-h time window is susceptible to interventions with NK1 antagonists that block the passage of aversive information from the dPAG to higher brain areas.

Inhibition of substance P-induced defensive behavior via neurokinin-1 receptor antagonism in the central and medial but not basolateral nuclei of the amygdala in male Wistar rats.

RATIONALE: The production of unconditioned defensive behaviors has been related to the amygdala, a key component of the encephalic aversion system. Microinjection of the neuropeptide substance P (SP) in the amygdala elicits defensive behaviors via the activation of type 1 neurokinin (NK-1) receptors. However, no studies have investigated whether intra-amygdala SP/NK-1 mechanisms can elicit other types of defensive responses, such as antinociception and ultrasonic vocalizations (USVs). METHODS: The present study investigated the effects of SP-induced activation of the neurokininergic system in three main nuclei of the amygdala-basolateral (BLA), central (CeA), and medial (MeA) nuclei-in rats that were subjected to the elevated plus maze (EPM), tail-flick test, and USV recording. The effects of SP in these amygdaloid nuclei were challenged with combined injections of the NK-1 receptor antagonist spantide. RESULTS: The present study showed that SP injections in the CeA and MeA but not BLA exerted anxiogenic-like effects. In contrast to the CeA, the anxiogenic-like effects of SP in the MeA were not dependent on NK-1 mechanisms. In the tail-flick test, SP microinjections produced antinociceptive effects only in the MeA through NK-1 receptor activation. No USV emissions were detected after the SP microinjections. CONCLUSIONS: The present study showed that NK-1 receptors in the CeA and MeA but not BLA are involved in defensive reactions to conditions of fear. The present results may provide a better understanding of the neurochemical mediation of fear states.

Angiogenic activity of latex from Euphorbia tirucalli Linnaeus 1753 (Plantae, Euphorbiaceae).

To assess the pro-angiogenic activity of Euphorbia tirucalli, commonly known as "avelós" plant, we performed a series of tests by applying an aqueous E. tirucalli latex solution (10 mg/mL) to the chorioallantoic membranes (CAMs) of 80 fertilized chicken eggs incubated in a temperature- and humidity-controlled automatic incubator. The results indicated that the aqueous latex solution increased vascular network formation compared to that with the negative control (p < 0.05) and the inhibitor control (p < 0.05). This suggests that under the experimental conditions tested, the aqueous latex solution induced an inflammatory response leading to neoangiogenesis.

Dorsal periaqueductal gray post-stimulation freezing is counteracted by neurokinin-1 receptor antagonism in the central nucleus of the amygdala in rats.

Electrical stimulation of the dorsal periaqueductal gray (dPAG) in rats generates defensive responses that are characterized by freezing and escape behaviors, followed by post-stimulation freezing that resembles symptoms of panic attacks. dPAG post-stimulation freezing involves the processing of ascending aversive information to prosencephalic centers, including the amygdala, which allows the animal to evaluate the consequences of stressful situations. The basolateral nucleus of the amygdala (BLA) is thought to act as a filter for innate and learned aversive information that is transmitted to higher structures. The central (CeA) and medial (MeA) nuclei of the amygdala constitute an output for the expression of fear reactions through projections to limbic and brainstem regions. Neurokinin (NK) receptors are abundant in the CeA, MeA, and BLA, but their role in the expression of defensive responses and processing of aversive information that is evoked by electrical stimulation of the dPAG is still unclear. In the present study, we examined the role of NK1 receptors in these amygdala nuclei in the expression of defensive responses induced by electrical stimulation of the dPAG in rats and fear memory of this aversive stimulation. Rats were implanted with an electrode into the dPAG for electrical stimulation and one cannula in the CeA, MeA, or BLA for injections of vehicle (phosphate-buffered saline) or the NK1 receptor antagonist spantide (SPA; 100 pmol/0.2 µl). Injections of SPA into the CeA but not BLA or MeA reduced the duration of post-stimulation freezing evoked by electrical stimulation of the dPAG, without changing the aversive thresholds of freezing or escape. Twenty-four hours later, exploratory behavior was evaluated in the elevated plus maze test (EPM) in the CeA group of rats. Electrical stimulation of the dPAG rats that received vehicle exhibited higher aversion to the open arms of the EPM than sham rats that did not receive any dPAG stimulation. SPA injections into the CeA prevented the proaversive effects of electrical stimulation of the dPAG assessed in the EPM 24 h later. The present results suggest that neurokininergic modulation via NK1 receptors in the CeA but not BLA or MeA is involved in the processing of aversive information derived from dPAG stimulation. The long-lasting consequences of electrical stimulation of the dPAG may be prevented by NK1 receptor antagonism in the CeA.

Medial prefrontal cortex serotonergic and GABAergic mechanisms modulate the expression of contextual fear: intratelencephalic pathways and differential involvement of cortical subregions.

Several lines of evidence indicate that the dorsal hippocampus (dH) and medial prefrontal cortex (mPFC) regulate contextual fear conditioning. The prelimbic (PrL), infralimbic (IL) and the anterior cingulate cortex (ACC) subregions of the mPFC likely play distinct roles in the expression of fear. Moreover, studies have highlighted the role of serotonin (5-hydroxytryptamine, 5-HT)- and γ-aminobutyric acid (GABA)-mediated mechanisms in the modulation of innate fear in the mPFC. The present study characterized dH-mPFC pathways and investigated the role of serotonergic and GABAergic mechanisms of the PrL, IL and ACC-area 1 (Cg1) in the elaboration of contextual fear conditioning using fear-potentiated startle (FPS) and freezing behavior in Rattus norvegicus. The results of neurotracing with microinjections of biotinylated dextran amine into the dH revealed a neural link of the dH with the PrL and ACC. Intra-PrL injections of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and the GABAA receptor-selective agonist muscimol reduced contextual FPS and freezing responses. Intra-Cg1 injections of muscimol but not 8-OH-DPAT decreased FPS and freezing responses. However, neither intra-IL injections of a 5-HT1A agonist nor of a GABAA agonist affected these defensive responses. Labeled neuronal fibers from the dH reached the superficial layers of the PrL cortex and spread to the inner layers of PrL and Cg1 cortices, supporting the pharmacological findings. The present results confirmed the involvement of PrL and Cg1 in the expression of FPS and freezing responses to aversive conditions. In addition, PrL serotoninergic mechanisms play a key role in contextual fear conditioning. This study suggests that PrL, IL and Cg1 distinctively contribute to the modulation of contextual fear conditioning.

Hormonal changes and increased anxiety-like behavior in a perimenopause-animal model induced by 4-vinylcyclohexene diepoxide (VCD) in female rats.

Perimenopause, a transition period that precedes menopause, is characterized by neuroendocrine, metabolic and behavioral changes, and is associated with increased vulnerability to affective disorders. The decrease in ovarian follicles during perimenopause contributes to a dynamic and complex hormonal milieu that is not yet well characterized. In rodents, 4-vinylcyclohexene diepoxide (VCD) induces a gradual depletion of ovarian follicles, modeling the transition to menopause in women. This study was aimed to investigate, in VCD-treated rats, the hormonal status and the behavior in the elevated plus-maze (EPM), a widely used test to assess anxiety-like behavior. From the postnatal day 28, rats were treated with VCD or vehicle for 15 days. At 80±5 days after the beginning of treatment the experiments were performed at proestrus and diestrus. In the first experiment rats were decapitated, ovary was collected and blood samples were taken for estradiol, progesterone, follicle stimulant hormone (FSH), testosterone, dihydrotestosterone (DHT) and corticosterone measurements. In the second experiment, rats were subjected to the EPM for 5 min, and behavioral categories recorded. Administration of VCD induced follicular depletion as well as an increase of the number of atretic follicles demonstrating the treatment efficacy. The transitional follicular depletion was accompanied by lower progesterone, testosterone and DHT with no changes in the FSH, estradiol and corticosterone plasma levels. On the EPM, rats showed decreased open arm exploration and increased risk assessment behavior, indicating increased anxiety. These findings show that administration of VCD to induce ovarian failure results in endocrine and anxiety-related changes that are similar to the symptoms exhibited by women during menopause transition. Thus, this model seems to be promising in the study of perimenopause-related changes.

Distinct effects of haloperidol in the mediation of conditioned fear in the mesolimbic system and processing of unconditioned aversive information in the inferior colliculus.

Chemical and electrical stimulation of the inferior colliculus (IC) causes defensive behavior. Electrical stimulation of the IC at the escape threshold enhances dopamine (DA) release in the prefrontal cortex. Intra-ventral tegmental area injections of quinpirole at doses that act presynaptically reduce the release of DA in the terminal fields of the mesolimbic system and clearly reduce conditioned fear in several animal models of anxiety. However, little is known about the involvement of DA in the mediation of unconditioned fear, such as the reactivity to acute stressors. The present study investigated the neural substrates mediated by DA transmission associated with emotional changes triggered by the activation or inhibition of D2 receptors during conditioned and unconditioned fear. We examined the effects of systemic or local injections of the DA-receptor antagonist and agonist haloperidol and quinpirole, respectively, into the IC in rats subjected to fear-potentiated startle, a Pavlovian paradigm that uses loud sounds as the unconditioned stimulus and light previously paired with footshock as the conditioned stimulus. We also assessed auditory-evoked potentials (AEPs) recorded from electrodes implanted in the IC. Intraperitoneal haloperidol administration dose-dependently enhanced AEPs induced by loud tones and inhibited fear-potentiated startle. Intra-IC injections of quinpirole left AEPs unchanged, suggesting that an optimal level of postsynaptic D2 receptors in the IC may regulate the transmission of aversive information through the midbrain tectum. These findings provide evidence of opposing DA-mediated mechanisms in fear/anxiety processes that depend on the area under study. The activity of the neural substrates of conditioned fear was attenuated by haloperidol, whereas midbrain neural substrates of unconditioned fear were enhanced. Thus, DA appears to regulate unconditioned fear at the midbrain level, likely by reducing the sensory gating of aversive events and reducing conditioned fear by acting at more rostral levels of the brain.

Participation of NK1 receptors of the amygdala on the processing of different types of fear.

The amygdala, medial hypothalamus, dorsal periaqueductal gray (dPAG), superior and inferior colliculus together constitutes the encephalic aversion system which has been considered the main neural substrate for the integration of unconditioned aversive behavioral states. Within the amygdala the basolateral nucleus (BLA) is thought to act as a filter for innate and learned aversive information to higher structures, whereas the central nucleus (CeA) is considered the main output for the expression of fear reactions through projections to limbic and brainstem regions. Although neurokinin (NK) receptors are abundant in the amygdala, their role in the processing and expression of fear is yet unclear. In this study, we examined the role of SP/NK1 receptor system of the CeA and BLA on the expression of defensive responses elaborated by Wistar rats submitted to elevated plus maze (EPM) and to electrical stimulation (ES) of the dPAG. For EPM test, cannulae were implanted in the CeA and BLA for injections of substance P (SP - 10 and 100pmol/0.2µL) and spantide (SPA - 10, 100 and 500pmol/0.2µL). For ES of dPAG, aversive thresholds for freezing and escape responses as well as post-stimulation freezing (PSF) were measured in rats treated with PBS and SPA (100pmol/0.2µL) in CeA. Injections of SP into the CeA, but not the BLA, produced anxiogenic-like effects in the EPM test. SPA injected into the CeA had no effect on the exploratory behavior of rats submitted to the EPM but blocked the effects of SP. The duration of dPAG-PSF was also reduced significantly following injection of SPA in CeA but had no effect on thresholds for freezing and escape responses. The EPM gives the animal a control over its environment i.e. the option to choose or not to enter into the open arm and dPAG-PSF is thought to reflect a period when the animal evaluates the significance of dPAG-evoked aversion once the unconditioned responses of freezing and escape were elicited. The data indicate that SP may be involved in mediating responses of the animal in only certain types of aversive behavior and suggests a differential participation of the NK1 receptors in the processing of distinct types of fear in the amygdala.

Glutamatergic mechanisms of the dorsal periaqueductal gray matter modulate the expression of conditioned freezing and fear-potentiated startle.

It is well known that excitatory amino acids induce unconditioned fear responses when locally injected into the dorsal periaqueductal gray matter (dPAG). However, there are only few studies about the involvement of excitatory amino acids mediation in dPAG in the expression of conditioned fear. The present series of experiments evaluates the participation of AMPA/Kainate and NMDA glutamatergic receptors of dPAG in the expression of conditioned fear, assessed by the fear-potentiated startle (FPS) and conditioned freezing responses. Wistar rats were subjected to fear conditioning to light. Twenty-four hours later, they received intra-dPAG injections of kainic acid or NMDA (AMPA/Kainate and NMDA agonists) and 1,2,3,4-Tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium salt hydrate (NBQX) or D(-)-2-Amino-7-phosphonoheptanoic acid (AP7) (AMPA/Kainate and NMDA antagonists) and were submitted to the FPS test. Conditioned freezing response was simultaneously measured. Effects of drug treatment on motor activity were evaluated in the open-field test. Intra-dPAG injections of glutamatergic agonists enhanced conditioned freezing and promoted pro-aversive effects in the FPS. Lower doses of the agonists had no effect or enhanced FPS whereas higher doses disrupted FPS, indicating a non-monotonic relationship between fear and FPS. The antagonist NBQX had no significant effects while AP7 decreased conditioned freezing but did not affect FPS. Both antagonists reduced the effects of the agonists. The obtained results cannot be attributed to motor deficits. The results suggest an important role of the AMPA/Kainate and NMDA mechanisms of the dPAG in the expression of conditioned freezing and FPS.

Involvement of midbrain tectum neurokinin-mediated mechanisms in fear and anxiety

Electrical stimulation of midbrain tectum structures, particularly the dorsal periaqueductal gray (dPAG) and inferior colliculus (IC), produces defensive responses, such as freezing and escape behavior. Freezing also ensues after termination of dPAG stimulation (post-stimulation freezing). These defensive reaction responses are critically mediated by γ-aminobutyric acid and 5-hydroxytryptamine mechanisms in the midbrain tectum. Neurokinins (NKs) also play a role in the mediation of dPAG stimulation-evoked fear, but how NK receptors are involved in the global processing and expression of fear at the level of the midbrain tectum is yet unclear. The present study investigated the role of NK-1 receptors in unconditioned defensive behavior induced by electrical stimulation of the dPAG and IC of male Wistar rats. Spantide (100 pmol/0.2 μL), a selective NK-1 antagonist, injected into these midbrain structures had anti-aversive effects on defensive responses and distress ultrasonic vocalizations induced by stimulation of the dPAG but not of the IC. Moreover, intra-dPAG injections of spantide did not influence post-stimulation freezing or alter exploratory behavior in rats subjected to the elevated plus maze. These results suggest that NK-1 receptors are mainly involved in the mediation of defensive behavior organized in the dPAG. Dorsal periaqueductal gray-evoked post-stimulation freezing was not affected by intra-dPAG injections of spantide, suggesting that NK-1-mediated mechanisms are only involved in the output mechanisms of defensive behavior and not involved in the processing of ascending aversive information from the dPAG.

Involvement of midbrain tectum neurokinin-mediated mechanisms in fear and anxiety.

Electrical stimulation of midbrain tectum structures, particularly the dorsal periaqueductal gray (dPAG) and inferior colliculus (IC), produces defensive responses, such as freezing and escape behavior. Freezing also ensues after termination of dPAG stimulation (post-stimulation freezing). These defensive reaction responses are critically mediated by γ-aminobutyric acid and 5-hydroxytryptamine mechanisms in the midbrain tectum. Neurokinins (NKs) also play a role in the mediation of dPAG stimulation-evoked fear, but how NK receptors are involved in the global processing and expression of fear at the level of the midbrain tectum is yet unclear. The present study investigated the role of NK-1 receptors in unconditioned defensive behavior induced by electrical stimulation of the dPAG and IC of male Wistar rats. Spantide (100 pmol/0.2 µL), a selective NK-1 antagonist, injected into these midbrain structures had anti-aversive effects on defensive responses and distress ultrasonic vocalizations induced by stimulation of the dPAG but not of the IC. Moreover, intra-dPAG injections of spantide did not influence post-stimulation freezing or alter exploratory behavior in rats subjected to the elevated plus maze. These results suggest that NK-1 receptors are mainly involved in the mediation of defensive behavior organized in the dPAG. Dorsal periaqueductal gray-evoked post-stimulation freezing was not affected by intra-dPAG injections of spantide, suggesting that NK-1-mediated mechanisms are only involved in the output mechanisms of defensive behavior and not involved in the processing of ascending aversive information from the dPAG.