African swine fever: A review of cleaning and disinfection procedures in commercial pig holdings.

African swine fever (ASF) is one of the most important diseases in pigs. Since there are no effective vaccines against the virus, farm biosecurity and good farming practices are the only effective tools to prevent the spread of the ASF virus (ASFV) in pig holdings. Hence, an important component of farm biosecurity is the Cleaning and Disinfection (C&D) procedure. Precise indications regarding the ideal disinfectant against ASFV are lacking, but every country has approved and/or authorized a list of biocides effective against ASFV. Lipidic solvents, which destroy the envelope of the virus and commercial disinfectants based on iodine and phenolic compounds are effective in inactivating the ASFV. This review describes the C&D protocol to apply in pig holdings with particular reference to ASFV.

Regulation of fear extinction versus other affective behaviors by discrete cortical scaffolding complexes associated with NR2B and PKA signaling.

In patients suffering from post-traumatic stress disorder (PTSD), fear evoked by trauma-related memories lasts long past the traumatic event and it is often complicated by general anxiety and depressed mood. This poses a treatment challenge, as drugs beneficial for some symptoms might exacerbate others. For example, in preclinical studies, antagonists of the NR2B subunit of N-methyl-d-aspartate receptors and activators of cAMP-dependent protein kinase (PKA) act as potent antidepressants and anxiolytics, but they block fear extinction. Using mice, we attempted to overcome this problem by interfering with individual NR2B and PKA signaling complexes organized by scaffolding proteins. We infused cell-permeable Tat peptides that displaced either NR2B from receptor for activated C kinase 1 (RACK1), or PKA from A-kinase anchor proteins (AKAPs) or microtubule-associated proteins (MAPs). The infusions were targeted to the retrosplenial cortex, an area involved in both fear extinction of remotely acquired memories and in mood regulation. Tat-RACK1 and Tat-AKAP enhanced fear extinction, all peptides reduced anxiety and none affected baseline depression-like behavior. However, disruption of PKA complexes distinctively interfered with the rapid antidepressant actions of the N-methyl-D-aspartate receptors antagonist MK-801 in that Tat-MAP2 blocked, whereas Tat-AKAP completely inverted the effect of MK-801 from antidepressant to depressant. These effects were unrelated to the MK-801-induced changes of brain-derived neurotrophic factor messenger RNA levels. Together, the findings suggest that NR2B-RACK1 complexes specifically contribute to fear extinction, and may provide a target for the treatment of PTSD. AKAP-PKA, on the other hand, appears to modulate fear extinction and antidepressant responses in opposite directions.

Gene expression patterns in the hippocampus and amygdala of endogenous depression and chronic stress models.

The etiology of depression is still poorly understood, but two major causative hypotheses have been put forth: the monoamine deficiency and the stress hypotheses of depression. We evaluate these hypotheses using animal models of endogenous depression and chronic stress. The endogenously depressed rat and its control strain were developed by bidirectional selective breeding from the Wistar-Kyoto (WKY) rat, an accepted model of major depressive disorder (MDD). The WKY More Immobile (WMI) substrain shows high immobility/despair-like behavior in the forced swim test (FST), while the control substrain, WKY Less Immobile (WLI), shows no depressive behavior in the FST. Chronic stress responses were investigated by using Brown Norway, Fischer 344, Lewis and WKY, genetically and behaviorally distinct strains of rats. Animals were either not stressed (NS) or exposed to chronic restraint stress (CRS). Genome-wide microarray analyses identified differentially expressed genes in hippocampi and amygdalae of the endogenous depression and the chronic stress models. No significant difference was observed in the expression of monoaminergic transmission-related genes in either model. Furthermore, very few genes showed overlapping changes in the WMI vs WLI and CRS vs NS comparisons, strongly suggesting divergence between endogenous depressive behavior- and chronic stress-related molecular mechanisms. Taken together, these results posit that although chronic stress may induce depressive behavior, its molecular underpinnings differ from those of endogenous depression in animals and possibly in humans, suggesting the need for different treatments. The identification of novel endogenous depression-related and chronic stress response genes suggests that unexplored molecular mechanisms could be targeted for the development of novel therapeutic agents.

High-resolution 3D MRI of mouse brain reveals small cerebral structures in vivo.

This work demonstrates technical approaches to high-quality magnetic resonance imaging (MRI) of small structures of the mouse brain in vivo. It turns out that excellent soft-tissue contrast requires the reduction of partial volume effects by using 3D MRI at high (isotropic) resolution with linear voxel dimensions of about 100-150 microm. The long T(2)* relaxation times at relatively low magnetic fields (2.35 T) offer the benefit of a small receiver bandwidth (increased signal-to-noise) at a moderate echo time which together with the small voxel size avoids visual susceptibility artifacts. For measuring times of 1-1.5 h both T(1)-weighted (FLASH) and T(2)-weighted (Fast Spin-Echo) 3D MRI acquisitions exhibit detailed anatomical insights in accordance with histological sections from a mouse brain atlas. Preliminary applications address the identification of neuroanatomical variations in different mouse strains and the use of Mn(2+) as a T(1) contrast agent for neuroaxonal tracing of fiber tracts within the mouse visual pathway.

Pharmacology and biology of corticotropin-releasing factor (CRF) receptors.

The biology of corticotropin-releasing factor (CRF) finds increasing interest in the scientific community because of the neuromodulatory actions of CRF on brain functions such as learning, anxiety, feeding, and locomotion. Additional actions on immunumodulation and apoptosis have recently been discovered. All actions of CRF are mediated by G protein-coupled receptors, which trigger different, sometimes opposite actions in different regions of the central nervous system. The CRF system exhibits considerable plasticity by the involvement of numerous different ligands, splice variants, and transductional couplings. The generation of multiple splice variants is facilitated by the intron exon structure of the CRF receptor genes.

Phosphorylated cAMP response element binding protein in the mouse brain after fear conditioning: relationship to Fos production.

Phosphorylation of the cAMP response element binding protein (pCREB) triggered by associative learning was monitored immunohistochemically in different areas of the mouse brain during a 6-h interval, starting immediately after training. One trial context-dependent fear conditioning was employed as a learning paradigm. Training consisted of contextual exposure followed by shock. Control groups consisted of naïve mice, mice exposed to the context alone and mice exposed to an immediate shock in the context. For all trained mice, the time course of CREB phosphorylation in hippocampus, parietal cortex and amygdaloid nuclei exhibited a biphasic pattern. The early phase was between 0 and 30 min, and the late phase was between 3 and 6 h after training. The animals exposed to context followed by an electric shock, as well as those exposed to an immediate electric shock, exhibited significantly higher pCREB levels than the mice subjected to context alone. During the late phase, the pCREB levels were highest in the mice exposed to the context followed by shock. It was observed that CREB phosphorylation and Fos production followed different regional and stimulus-dependent patterns. It is suggested that the early phase of pCREB increase may be related to stress-related behaviors, whereas the late phase may rather relate to memory consolidation.

Pharmacological and chemical properties of astressin, antisauvagine-30 and alpha-helCRF: significance for behavioral experiments.

Corticotropin releasing factor (CRF) represents an early chemical signal in the stress response and modulates various brain functions through G protein-coupled receptors. Two CRF receptor subtypes, CRF(1) and CRF(2), have been identified. Since the physicochemical properties of CRF receptor antagonists might influence their biological potency, the peptidic antagonists astressin, alpha-helical CRF(9-41) (alpha-helCRF) and antisauvagine-30 (aSvg-30) have been analyzed. The rank order of solubility of these compounds in artificial cerebrospinal fluid (aCSF, pH 7.4) was aSvg-30>alpha-helCRF>>astressin, whereas the rank order of relative lipophilicity as determined with RP-HPLC was alpha-helCRF>astressin>aSvg-30. The calculated isoelectric points were 4.1 (alpha-helCRF), 7.4 (astressin) and 10.0 (aSvg-30). According to Schild analysis of the CRF receptor-dependent cAMP production of transfected HEK cells, aSvg-30 exhibited a competitive antagonism and displayed a 340 fold selectivity for mCRF(2 beta) receptor. For astressin, however, the pharmacodynamic profile could not be explained by a simple competitive mechanism as indicated by Schild slopes >1 for rCRF(1) or mCRF(2 beta) receptor. Behavioral experiments demonstrated that after i.c.v. injection, alpha-helCRF reduced oCRF-induced anxiety-like behavior in the elevated plus-maze, whereas astressin, despite its higher in vitro potency, did not. These findings could be explained by different physicochemical properties of the antagonists employed.

A single amino acid serves as an affinity switch between the receptor and the binding protein of corticotropin-releasing factor: implications for the design of agonists and antagonists.

In view of the observation that corticotropin-releasing factor (CRF) affects several brain functions through at least two subtypes of G protein-dependent receptors and a binding protein (CRFBP), we have developed synthetic strategies to provide enhanced binding specificity. Human/rat CRF (h/rCRF) and the CRF-like peptide sauvagine (Svg), differing in their affinities to CRFBP by two orders of magnitude, were used to identify the residues determining binding to CRFBP. By amino acid exchanges, it was found that Ala(22) of h/rCRF was responsible for this peptide's high affinity to CRFBP, whereas Glu(21) located in the equivalent position of Svg prevented high affinity binding to CRFBP. Accordingly, [Glu(22)]h/rCRF was not bound with high affinity to CRFBP in contrast to [Ala(21)]Svg, which exhibited such high affinity. Furthermore, the affinity of both peptides to either CRF receptor (CRFR) subtype was not reduced by these replacements, and their subtype preference was not changed. Thus, exchange of Ala and Glu and vice versa in positions 22 and 21 of h/rCRF and Svg, respectively, serves as a switch discriminating between CRFBP and CRFR. On the basis of this switch function, development of new specific CRF agonists and antagonists is expected to be facilitated. One application was the modification of the CRF antagonist astressin (Ast), whose employment in animal experiments is limited by its low solubility in cerebrospinal fluid. Introduction of Glu residues into Ast generated with [Glu(11,16)]Ast an acidic astressin, which efficiently antagonized in vivo the CRFR1-dependent reduction of locomotion induced by ovine CRF without detectable binding to CRFBP.

Behavior and severity of adjuvant arthritis in four rat strains.

Previous research has suggested that behavioral traits of the histocompatible Lewis and Fischer strains of rats could be related to the difference in their susceptibility to adjuvant arthritis (AA). In the present study, the predictive value of behavioral markers in susceptibility to AA was investigated in nonhistocompatible inbred DA, Lewis, Albino Oxford (AO), and outbred Wistar strain. Behavioral profiles (open filed test and forced swim test) were determined prior to immunization with a single intradermal injection of complete Freund's adjuvant. Animals were daily scored for clinical signs of AA. The occurrence of certain behaviors and clinical indices of AA was significantly associated with strain membership. Discriminant analysis identified strain-related behavioral and illness profiles with very few overlaps among the phenotypes. Discriminant classification significantly exceeded the proportion of cases, which could have been correctly classified on the basis of chance. Open field behavior, in particular, exploration and grooming, differentiated among AA-susceptible and AA-resistant strains. Multiple regression analysis indicated that severity of AA (maximum clinical sign) can be predicted by the latency time and grooming behavior in the open field independently of strain membership. No clear distinction between AA-susceptible and AA-resistant strains was found with respect to forced swim test immobility. It was concluded that (a) strain-related genetic predisposition is important for the expression of certain behavioral traits and for susceptibility to AA and (b) open field behaviors, particularly grooming and latency, predict susceptibility to AA across different rat strains.

Modulation of humoral immune responses in the rat by centrally applied Met-Enk and opioid receptor antagonists: functional interactions of brain OP1, OP2 and OP3 receptors.

We have previously demonstrated that central application of leucine-enkephalin (Leu-Enk) elicits potentiation and suppression of humoral immune responses through OP(1) (delta) and OP(2) (kappa) receptors, respectively. Interestingly, both effects were found to be additionally dependent on OP(3) (mu) receptor function. In the present study, we have further investigated whether opioid receptor interactions underlie the immunomodulatory effects of endogenous opioids as well as exogenously applied methionine-enkephalin (Met-Enk). For that purpose, the plaque-forming cell (PFC) response was determined in rats injected intracerebroventricularly (i.c.v.) with opioid receptor-selective antagonists and Met-Enk. Application of the OP(1) antagonist ICI 174864, but not naltrindole, resulted in suppression of the PFC response. In contrast, i.c.v. injection of the OP(2) selective antagonist nor-binaltorphimine (nor-BNI) significantly potentiated the PFC response. Both effects, presumably mediated by endogenous opioid peptides, were antagonized by the OP(3) receptor antagonist beta-funaltrexamine (beta-FNA) at a dose that was devoid of immunomodulatory activity. The immunopotentiation of the PFC response induced by Met-Enk was reversed by OP(1) receptor antagonists, naltrindole and ICI 174864, but not by beta-FNA or nor-BNI. On the basis of these and previous findings, it may be concluded that central OP(3) receptors are permissive for the central immunomodulatory action of endogenous opioid peptides and Leu-Enk. In contrast, the central immunoenhancing effect of Met-Enk appears to be mediated through OP(3)-independent OP(1) receptors.

Role of regional neurotransmitter receptors in corticotropin-releasing factor (CRF)-mediated modulation of fear conditioning.

We have demonstrated previously that stimulation of hippocampal corticotropin-releasing factor (CRF) receptors enhances, whereas stimulation of CRF receptors in the lateral intermediate septum impairs learning, as indicated by fear conditioning. Here, we report that the action of CRF within the hippocampus and lateral septum require muscarinic and D2 receptors, respectively.

Deletion of crhr2 reveals an anxiolytic role for corticotropin-releasing hormone receptor-2.

Corticotropin-releasing hormone (Crh), a 41-residue polypeptide, activates two G-protein-coupled receptors, Crhr1 and Crhr2, causing (among other transductional events) phosphorylation of the transcription factor Creb. The physiologic role of these receptors is only partially understood. Here we report that male, but not female, Crhr2-deficient mice exhibit enhanced anxious behaviour in several tests of anxiety in contrast to mice lacking Crhr1. The enhanced anxiety of Crhr2-deficient mice is not due to changes in hypothalamic-pituitary-adrenal (HPA) axis activity, but rather reflects impaired responses in specific brain regions involved in emotional and autonomic function, as monitored by a reduction of Creb phosphorylation in male, but not female, Crhr2-/- mice. We propose that Crhr2 predominantly mediates a central anxiolytic response, opposing the general anxiogenic effect of Crh mediated by Crhr1. Neither male nor female Crhr2-deficient mice show alterations of baseline feeding behaviour. Both respond with increased edema formation in response to thermal exposure, however, indicating that in contrast to its central role in anxiety, the peripheral role of Crhr2 in vascular permeability is independent of gender.

In vivo NMDA/dopamine interaction resulting in Fos production in the limbic system and basal ganglia of the mouse brain.

Glutamatergic and dopaminergic effects on molecular processes have been extensively investigated in the basal ganglia. It has been demonstrated that NMDA and dopamine D(1) and D(2) receptors interact in the regulation of signal transduction and induction of transcription factors. In the present experiments, NMDA/dopamine interactions were investigated in the normosensitive caudate nucleus, hippocampus and amygdala by monitoring Fos production. We demonstrated that NMDA and the D(1) receptor agonist SKF 38393 triggered Fos levels in a distinct, non-overlapping and region-specific pattern. NMDA injected intraperitoneally (i.p.) elevated Fos levels in all hippocampal subfields and the central amygdala, whereas SKF 38393 triggered Fos production in basomedial, cortical, medial amygdala and caudate nucleus. The NMDA receptor antagonist CGS 19755 prevented NMDA- and SKF 38393-triggered Fos production in all investigated brain areas. Similarly, the D(1) receptor antagonist SCH 23390 inhibited the effects produced by SKF 38393 or NMDA. The D(2) receptor antagonist sulpiride exerted synergistic and antagonistic effects on NMDA- and SKF 38393-triggered Fos production, in a region specific manner. These data suggest that NMDA and dopamine receptors regulate Fos production within the limbic system and basal ganglia through regionally differentiated but interdependent actions.

Actions of CRF and its analogs.

Corticotropin-releasing factor (CRF), urocortin, sauvagine and urotensin I form the CRF family. These peptides bind with different affinities to two subtypes of CRF receptor (CRFR), CRFR1 and CRFR2. The latter exists as two splice variants, the neuronal CRFR2a and the peripheral CRFR2b. CRFR is a G protein-dependent receptor which acts mainly through Gs enhancing cAMP production. However, CRFR1 expressed in neutrophils of the spleen in response to immunologic stimulation and psychological stress does not seem to function through Gs, as indicated by the inability of CRF to stimulate the cAMP production of CRFR1+ neutrophils. Besides the two receptors, a 37 kD CRF binding protein (CRF-BP) binds several CRF peptides with high affinity. CRFR and CRF-BP do not share a common amino acid sequence representing the ligand binding site. In view of the unusually slow offrate of CRF-BP, it is proposed that CRF-BP provides an efficient uptake of free extracellular CRF. Thus, the time of exposure of CRFR to CRF or urocortin can be limited. At this time, the fate of the ligand CRF-BP complex is unclear. CRFR1 is not only involved in the hypophyseal stimulation of corticotropin release, but hippocampal CRFR1 mediates enhancement of stress-induced learning. CRFR1 may also be involved in basic anxiety. In contrast, at least in the mouse, CRFR2 of the lateral intermediate septum mediates tonic impairment of learning. In response to stressful stimuli or after local injection of high CRF doses, CRFR2 mediates anxiety. Effects requiring CRFR2 can be blocked specifically by the recently developed peptidic antagonist antisauvagine-30.

Differential impairment of auditory and contextual fear conditioning by protein synthesis inhibition in C57BL/6N mice.

A 1-trial fear conditioning was used to investigate the temporal development of fear responses expressed as increase of freezing or heart rate and its impairment by the protein synthesis inhibitor cycloheximide (CHX) in male C57BL/6N mice. Heart rate was measured with an implanted transmitter. In the memory tests, mice were exposed to tone and context provided either as foreground or background stimulus during training. The fear responses developed differently from 0 to 24 hr after training under these 3 conditions. A single pretraining CHX injection impaired both memory forms, whereas a single posttraining CHX injection impaired tone- but not context-dependent memory, with the context provided as background stimulus. It was concluded that consolidation of tone-, foreground context-, and background context-dependent fear conditioning may be mediated by partly different neuronal or partly different biochemical pathways, or both.

Modulation of learning and anxiety by corticotropin-releasing factor (CRF) and stress: differential roles of CRF receptors 1 and 2.

The differential modulation of learning and anxiety by corticotropin-releasing factor (CRF) through CRF receptor subtypes 1 (CRFR1) and 2 (CRFR2) is demonstrated. As learning paradigm, context- and tone-dependent fear conditioning of the mouse was used. Injection of CRF into the dorsal hippocampus before training enhanced learning through CRFR1 as demonstrated by the finding that this effect was prevented by the local injection of the unselective CRFR antagonist astressin, but not by the CRFR2-specific antagonist antisauvagine-30 (anti-Svg-30). In contrast, injection of CRF into the lateral intermediate septum impaired learning through CRFR2, as demonstrated by the ability of antisauvagine-30 to block this effect. When antisauvagine-30 was injected alone into the lateral intermediate septum, learning was enhanced. Such tonic control of learning was not observed when astressin or antisauvagine-30 was injected into the dorsal hippocampus. Injection of CRF after the training into the dorsal hippocampus and the lateral intermediate septum also enhanced and impaired learning, respectively. Thus, it was indicated that CRF acted on memory consolidation. It was concluded that the observed effects reflected changes of associative learning and not arousal, attention, or motivation. Although a dose of 20 pmol human/rat CRF was sufficient to affect learning significantly, a fivefold higher dose was required to induce anxiety by injection into the septum. Immobilization for 1 hr generated a stress response that included the induction of anxiety through septal CRFR2 and the subsequent enhancement of learning through hippocampal CRFR1. The involvement of either receptor subtype was demonstrated by region-specific injections of astressin and antisauvagine-30.

Corticotropin-releasing factor receptor 1 in mouse spleen: expression after immune stimulation and identification of receptor-bearing cells.

A specific polyclonal Ab against the N-terminal domain of corticotropin-releasing factor (CRF) receptor, type 1 (CRF-R1), was employed to an immunohistochemical analysis of the spleen from naive mice and mice exposed to an immune challenge. Cell types stained with anti-CRF-R1 Ab were identified by their nuclear shapes and colocalization with the cell type-specific markers ER-MP58, ER-MP20, Moma-1, Moma 2, anti-CD3e mAbs, and anti-Ig Ab. Only a few clusters of CRF-R1+ cells were found in spleen sections of naive mice at sites typical for granulopoietic islands. However, a 17-fold increase in the mean number of CRF-R1+ cells was noted within hours following a challenge of acute systemic inflammation induced by i.p. administration of LPS. The majority of these cells were identified as mature neutrophils. CRF-R1 was shown to mediate suppression of the IL-1beta secretion by these cells. However, at later time points a large number of granulocyte-macrophage precursors was strongly labeled with anti-CRF-R1 Ab. Western blot analysis of splenic membranes from animals treated with LPS revealed a m.w. of approximately 70,000 for CRF-R1. Subcellular staining patterns were suggestive for the predominant localization of CRF-R1 on granule membranes. CRF-R1 mRNA was detected in spleen but not in bone marrow and peripheral blood leukocytes from naive mice. Thus, it was indicated that CRF-R1 was not produced constitutively by mature or immature neutrophils. Its production was rather triggered by inflammatory stimuli.

Strain and substrain differences in context- and tone-dependent fear conditioning of inbred mice.

The performance of C57BL/6J (6J), C57BL/6N (6N), DBA/2J (2J) and DBA/2N (2N) mice in context- and tone-dependent fear conditioning was determined 24 h after fear conditioning to evaluate and compare different behavioral measures as indices of emotional learning. Freezing, the change in activity and the size of the explored area were evaluated as behavioral parameters indicating fear. Additionally, the heart rate (HR) increase elicited by tone presentation was evaluated as an autonomic indicator of fear. During the context-dependent memory test, freezing was high only in 6J and 6N mice, whereas a drop of activity and a reduced exploratory area was measured in all strains. During the tone-dependent memory test, high freezing, low activity, reduced exploratory area and a strong HR increase were demonstrated only in 6N and 6J mice, whereas behavioral and HR changes of 2J and 2N mice were always low. In extinction tests, context- and tone-dependent freezing of 6J mice decayed significantly faster than the freezing of 6N mice, whereas in both substrains the conditioned tachycardia to tone extinguished similarly in the home cage. The data demonstrate that monitoring of additional behavioral measures besides freezing and autonomic measures is necessary to interpret differences in associative learning performance of mouse strains that could be related to a differential expression of fear.

Characterization of native corticotropin-releasing factor receptor type 1 (CRFR1) in the rat and mouse central nervous system.

Corticotropin releasing factor (CRF), the most important regulator of various responses to stress, acts through CRF receptors (CRFR). For their characterization in brain tissue of Sprague-Dawley rats and C57BL/6J mice, a recently described polyclonal antibody directed against the N-terminus of rat CRFR1 (rCRFR1) was used. The molecular weights of rat and mouse brain receptors were determined by Western blot analysis to be 80,000-76,000 and 83,000-79,000, respectively, whereas molecular weights of 72,000-59,000 were observed for CRFR1 from rat and mouse pituitary. Immunohistochemical analysis was performed with brain sections of naive rats and mice. Strong CRFR1 staining was detected in the cortex, cerebellum, mesencephalon and pons of both species, whereas weak staining was observed in amygdala and hippocampus. The striatum did not show immunoreactivity. The density of immunostaining was significantly lower in murine than in rat cortex. In contrast, in the pons and mesencephalon of mice, higher density of immunostaining was observed than in the same brain structures of rats. On the basis of the observed differences, it is suggested that CRFR1 is differentially processed in rats and mice. In addition, the density of CRFR1 staining differed between both species.