Ethanol Consumption by Fawn-Hooded Rats Following Abstinence Effect of Naltrexone and Changes in μ-Opioid Receptor Density

BACKGROUND: Relapse after abstinence can be modelled in rats using an alcohol deprivation effect (ADE) of enhanced ethanol consumption after a period of enforced abstinence from ethanol; however, not all rat strains display such an effect. We wanted to examine the effect of naltrexone on ethanol consumption by ethanol-preferring Fawn-Hooded (FH) rats using such a model. METHODS: FH rats were given continual free-choice access to a 5% ethanol solution or water (4 weeks) followed by 2 weeks of water alone. At the end of this abstinence period, osmotic minipumps were implanted subcutaneously to deliver saline (n = 4) or naltrexone (n = 4; 8.4 mg/kg/day for 4 weeks). After recovery from surgery, the rats were again given access to 5% ethanol under the same free-choice conditions (4 weeks). A third group of age-matched controls drank only water during the behavioral trial. At the end of the behavioral trial, the rats were decapitated and an autoradiographic examination was made of micro-opioid receptor density through the forebrain using the ligand [125I]FK-33824. RESULTS: First, a period of enforced abstinence from ethanol consumption caused a significant (p < 0.05) and prolonged increase in ethanol preference (+18%) and decrease in water consumption (-53%), although the volume of ethanol consumed (ml/day) did not vary, indicating an atypical ADE in this rat strain. Second, naltrexone significantly (p < 0.05) decreased ethanol consumption by the FH rats in terms of absolute amount of ethanol consumed and preference for ethanol solution, but this effect of naltrexone diminished over time, concurrent with a robust and significant elevation in micro-opioid receptor density in all brain regions examined (p < 0.05). Finally, ethanol consumption alone also upregulated micro-opioid receptor density relative to nondrinking controls in a number of brain regions, which included the nucleus accumbens (+29%) and caudate-putamen (+15%,p < 0.05), but decreased micro-opioid receptor density in other regions including the substantia nigra pars reticulata, which was suggestive of an indirect effect on micro-opioid receptors. CONCLUSIONS: The data suggest that continual long-term naltrexone treatment may not be effective in the treatment of alcoholism, possibly because of the induced increase in micro-opioid receptor density.     

Acute and chronic restraint stress: effects on [I]-galanin binding in normotensive and hypertensive rat brain

The neuropeptide galanin (GAL) has been implicated in the neural response to a number of stressors including restraint; however, the effect of restraint stress on GAL receptor density in the central nervous system (CNS) has not been investigated. Normotensive (Wistar-Kyoto; WKY) and hypertensive (spontaneously hypertensive; SHR) rats were subjected to a daily 60-min restraint stress paradigm for 0 (control), 1, 3, 5 or 10 consecutive days, and the density of [¹²⁵I]-GAL binding sites following exposure to restraint was compared between strains using quantitative autoradiography. Significant differences in basal (no stress) levels of GAL receptor density between WKY and SHR were detected in regions such as the central nucleus of the amygdala (Ce) and ventromedial hypothalamus (VMH) (P<0.05). In WKY, restraint stress (1 day) induced significant decreases in GAL receptor density in forebrain regions such as the Ce (−41%) and medial nucleus of the amygdala (−41%) (P<0.05). Chronic restraint (10 days) did not induce significant decreases in these nuclei in WKY, indicating that forebrain neurons containing GAL receptors in WKY possessed a functional ability to adapt to repeated restraint. In addition, restraint stress induced significant decreases in GAL receptor density in SHR in regions such as the lateral parabrachial nucleus (−43%; 5 days of restraint) and hypoglossal nucleus (−18% for entire restraint period) (P<0.05). In conclusion, restraint stress resulted in region- and strain-specific alterations in GAL receptor density, some of which may contribute to the altered stress response previously observed in hypertensive rats. The results clearly support the hypothesis that neuropeptides such as GAL are an integral component of the neural response to psychological stress, although the functional significance of the changes in GAL receptor density described in this study awaits elucidation.     

Sigma-1 receptor ligand PRE-084 reduced infarct volume, neurological deficits, pro-inflammatory cytokines and enhanced anti-inflammatory cytokines after embolic stroke in rats

Sigma receptor agonists have been found to provide potent neuroprotection in rats and mice. This neuroprotection is thought to be mediated through anti-excitotoxic mechanisms. Neuroprotective and immune modulatory effects of sigma ligands have not been investigated in embolic stroke. In the present study, rats were subjected to embolic stroke or sham stroke and were treated with the sigma-1 receptor agonist PRE-084 (5mg/kg i.p.) or saline vehicle 3 and 24h after stroke. Infarct volume and behavioural tests were conducted, and cytokine levels (ILs-1α and β, IL-2, IL-4, IL-6, IL-10, GM-CSF and TNF-α) were determined in ischemic and non-ischemic cortices. Axonal damage was determined using the pNF-H ELISA assay. Treatment with PRE-084 afforded neuroprotection following embolic stroke as evidenced by significantly reduced infarct volume and improved behavioural outcome. Remarkably, treatment with PRE-084 reduced levels of pro-inflammatory cytokines and enhanced anti-inflammatory cytokines. Levels of pNF-H were lower in rats treated with PRE-084 suggesting reduced axonal damage but this finding did not reach statistical significance. The findings of the present study suggest that part of the neuroprotective effects of sigma-1 receptor agonists may be mediated through a dual effect on cytokine release following stroke.     

Comparison of neuropeptide Y immunoreactivity in hypothalamic and brainstem nuclei of young and mature spontaneously hypertensive and normotensive Wistar-Kyoto rats

The concentration of neuropeptide Y immunoreactivity (NPY-ir) was measured in 8 hypothalamic and 5 brainstem nuclei of 6- and 14-week-old spontaneously hypertensive (SH) and Wistar-Kyoto (WKY) rats. Strain differences were observed in 3 hypothalamic nuclei and age-dependent changes occurred in 3 hypothalamic and 2 brainstem nuclei. In both the ventromedial hypothalamic nucleus and locus coeruleus the observed change in NPY-ir with age in SH rats was significantly different to the change observed in the WKY. These strain- and age-related differences in NPY-ir may be of relevance in the development of hypertension in the SH rat.     

Minocycline treatment attenuates microglia activation and non-angiotensin II [125I] CGP42112 binding in brainstem following nodose ganglionectomy

We have previously shown that following unilateral nodose ganglionectomy, [125I] CGP42112 binds to a non-angiotensin II (Ang II) related binding site in rat dorsal motor nucleus of the vagus nerve, ambiguus nucleus and nucleus of the solitary tract. Furthermore, this up-regulated binding site localizes with activated microglia. Given that some tetracyclines may inhibit microglia activation in brain, we examined the effect of minocycline treatment on the binding of [125I] CGP42112 and [3H] PK11195 (an established radioligand for microglia), as well as OX-42 immunoreactivity (an immunomarker for activated microglia), following nodose ganglionectomy. Male Wistar Kyoto rats underwent unilateral nodose ganglionectomy or sham operation and were treated with saline or minocycline (50 mg/kg i.p.) 12 h before surgery and twice daily after surgery (each 50mg/kg i.p.) for 3 days. Subsequent to nodose ganglionectomy, [125I] CGP42112 binding (insensitive to PD123319 or Ang II) was increased approximately two-fold in the ipsilateral nucleus of the solitary tract and was also induced in the ipsilateral dorsal motor nucleus of the vagus nerve and ambiguus nucleus of saline-treated rats. Treatment with minocycline reduced this non-angiotensin II [125I] CGP42112 binding (40-50% reduction) in the nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve and ambiguus nucleus. Analogous experiments using [3H] PK11195 also revealed up-regulated binding in the ipsilateral nucleus of the solitary tract ( approximately 205%), dorsal motor nucleus of the vagus nerve (approximately 80%) and ambiguus nucleus (approximately 210%) of saline-treated rats following nodose ganglionectomy, which was reduced by 40-100% with minocycline treatment. Immunoreactivity to OX-42 confirmed an increase in microglia activation and accumulation of macrophages in these brain stem nuclei following nodose ganglionectomy, which was also attenuated following treatment with minocycline. These data demonstrate that non-Ang II [125I] CGP42112 binding following nodose ganglionectomy is attenuated by minocycline treatment. This minocycline-induced effect was associated with reduced activation of microglia and an apparent reduction in the number of macrophages in the abovementioned nuclei. This evidence suggests that a non-Ang II [125I] CGP42112 binding site is located on, or associated with, activated microglia and macrophages, providing a useful tool with which to quantitate the neuroprotective effects of centrally acting anti-inflammatory compounds.     

Localization of AT(2) receptors in the nucleus of the solitary tract of spontaneously hypertensive and Wistar Kyoto rats using [125I] CGP42112: upregulation of a non-angiotensin II binding site following unilateral nodose ganglionectomy

We have examined the binding distribution of a selective AT(2) receptor ligand [125I] CGP42112 in the brain of adult Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). AT(2) receptor localization was also examined in the rat brainstem following unilateral nodose ganglionectomy. Specific [125I] CGP42112 binding was observed in discrete brain regions from both rat strains, including the nucleus of the solitary tract (NTS), and did not differ between WKY and SHR. [125I] CGP42112 binding in the NTS revealed an AT(2) receptor component that was displaceable by PD 123319 and Ang II (50-58%), as well as a non-angiotensin II receptor component (42-49%). Following unilateral nodose ganglionectomy, [125I] CGP42112 binding density on the denervated side of the NTS was increased approximately two-fold in both WKY and SHR. This increased [125I] CGP42112 binding density in the ipsilateral NTS was comprised of a greater non-angiotensin II component than that observed in the sham groups, since only approximately 30% was displaced by PD123319 and angiotensin II. Furthermore, [125I] CGP42112 also revealed high binding density on the denervated side in the dorsal motor nucleus and the nucleus ambiguus in both WKY and SHR. AT(2) receptor immunoreactivity was also visualised in the NTS of sham operated rats, but was not observed in the dorsal motor nucleus or the nucleus ambiguus, nor was it up-regulated following nodose ganglionectomy. These results demonstrate, for the first time, an AT(2) receptor binding site in the NTS, as well as a non-angiotensin II [125I] CGP42112 binding site. These studies also demonstrate that nodose ganglionectomy represents a useful model in which to study a non-angiotensin II [125I] CGP42112 binding site that is up-regulated following degeneration of afferent vagal nerves.     

Acute and chronic restraint stress: effects on [125I]-galanin binding in normotensive and hypertensive rat brain

The neuropeptide galanin (GAL) has been implicated in the neural response to a number of stressors including restraint; however, the effect of restraint stress on GAL receptor density in the central nervous system (CNS) has not been investigated. Normotensive (Wistar-Kyoto; WKY) and hypertensive (spontaneously hypertensive; SHR) rats were subjected to a daily 60-min restraint stress paradigm for 0 (control), 1, 3, 5 or 10 consecutive days, and the density of [125I]-GAL binding sites following exposure to restraint was compared between strains using quantitative autoradiography. Significant differences in basal (no stress) levels of GAL receptor density between WKY and SHR were detected in regions such as the central nucleus of the amygdala (Ce) and ventromedial hypothalamus (VMH) (P<0.05). In WKY, restraint stress (1 day) induced significant decreases in GAL receptor density in forebrain regions such as the Ce (-41%) and medial nucleus of the amygdala (-41%) (P<0.05). Chronic restraint (10 days) did not induce significant decreases in these nuclei in WKY, indicating that forebrain neurons containing GAL receptors in WKY possessed a functional ability to adapt to repeated restraint. In addition, restraint stress induced significant decreases in GAL receptor density in SHR in regions such as the lateral parabrachial nucleus (-43%; 5 days of restraint) and hypoglossal nucleus ( approximately -18% for entire restraint period) (P<0.05). In conclusion, restraint stress resulted in region- and strain-specific alterations in GAL receptor density, some of which may contribute to the altered stress response previously observed in hypertensive rats. The results clearly support the hypothesis that neuropeptides such as GAL are an integral component of the neural response to psychological stress, although the functional significance of the changes in GAL receptor density described in this study awaits elucidation.     

Regional brain concentrations of calcitonin gene-related peptide in spontaneously hypertensive and normotensive Wistar-Kyoto rats

The regional brain and spinal cord concentrations of calcitonin gene-related peptide (CGRP) were measured in age-matched (22-23-week-old) spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. The highest concentration of CGRP in the WKY rats was in the spinal cord (172 +/- 9 pmol/g), followed by the medulla oblongata/pons (88 +/- 5 pmol/g). The relative order of distribution in the remaining regions was: hypothalamus (12.6 +/- 0.8 pmol/g) = striatum greater than thalamus greater than midbrain = hippocampus greater than cortex (2.1 +/- 0.3 pmol/g). The concentration of CGRP in the cerebellum was at the level of the assay's sensitivity (0.5 pmol/g). The relative order of distribution in the SHR strain was essentially the same. However, in comparison with the WKY rats, the SHR had significantly lower levels of CGRP in the hippocampus (-47%), striatum (-49%) and medulla oblongata/pons (-24%), and in the spinal cord (-24%). In younger age-matched (16-17-week-old) rats, the spinal cord and medulla oblongata/pons concentrations of CGRP were also lower in SHR than in WKY rats. CGRP is a putative neurotransmitter which, when administered centrally or peripherally, has potent cardiovascular effects. The reduced levels of this peptide may be an important factor in the cardiovascular and/or behavioural abnormalities of the SHR strain.     

The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated bacterial clumping

Aggregation and biofilm formation are critical mechanisms for bacterial resistance to host immune factors and antibiotics. Autotransporter (AT) proteins, which represent the largest group of outer-membrane and secreted proteins in Gram-negative bacteria, contribute significantly to these phenotypes. Despite their abundance and role in bacterial pathogenesis, most AT proteins have not been structurally characterized, and there is a paucity of detailed information with regard to their mode of action. Here we report the structure–function relationships of Antigen 43 (Ag43a), a prototypic self-associating AT protein from uropathogenic Escherichia coli. The functional domain of Ag43a displays a twisted L-shaped β-helical structure firmly stabilized by a 3D hydrogen-bonded scaffold. Notably, the distinctive Ag43a L shape facilitates self-association and cell aggregation. Combining all our data, we define a molecular “Velcro-like” mechanism of AT-mediated bacterial clumping, which can be tailored to fit different bacterial lifestyles such as the formation of biofilms.Autotransporter (AT) proteins represent a large family of diverse secreted and outer-membrane proteins present in numerous Gram-negative bacteria, including all classes of Proteobacteria as well as numerous species of Chlamydiales and Fusobacteria (1–4). They are a core component of a molecular mechanism for the delivery of cargo protein domains across the outer membrane, and are often associated with important biological functions such as adhesion, aggregation, toxicity, and biofilm formation. All AT proteins are defined by a specific domain architecture that comprises an N-terminal signal sequence that directs secretion of the protein across the inner membrane via the general secretory pathway, a passenger (α) domain that is either anchored to the cell surface or released into the external milieu and determines the functional characteristics of the protein, and a translocation (β) domain that inserts into the outer membrane (5). Originally, AT proteins were thought to possess structural properties that facilitate their independent transport across the bacterial membrane and final routing to the cell surface (3). However, it is now known that accessory factors such as components of the Bam complex, periplasmic chaperones (SurA, Skp, DegP, and FkpA), and the translocation and assembly module contribute to the secretion of some AT proteins (6, 7).The AIDA-I–type AT proteins represent the largest and most diverse group of the AT superfamily (8). In Escherichia coli, 16 AIDA-I–type subgroups have been identified, which differ in the length and sequence of their passenger domain and their functional properties (8). Although these AT proteins show substantial sequence and functional variability, they are predicted to adopt common domain architectures; the transporter β-domain folds into the canonical β-barrel, and the functional passenger domain generally incorporates a right-handed β-helix (9).Antigen 43 (Ag43) is one of the best-studied AIDA-I–type AT proteins. This phase-variable AT protein is produced as a 1,039-amino acid preprotein incorporating an N-terminal signal peptide that directs translocation across the cytoplasmic membrane into the periplasm and a classical passenger (α⁴³)–translocation (β⁴³) domain structure (Fig. 1A). Processing of Ag43 occurs between the functional α⁴³ and the translocating β⁴³ domains; however, the two subunits remain in contact via noncovalent interactions (3), with the α⁴³ domain protruding ∼10 nm from the cell surface, where it is free to interact with Ag43 passenger domains presented on the surface of neighboring cells.Open in a separate windowFig. 1.Domain organization and structure of the Ag43a passenger domain (α^43a). (A) Schematic showing the arrangement of the protein domains: signal peptide (SP), passenger domain (α-domain), autochaperone domain (AC), and translocator domain (β-domain). (B) Ribbon representation of the secondary-structure elements of the α^43a β-helix with β-strands and loops colored in blue and green, respectively. Loop 1 and loop 2 protruding from the β-spine are colored in red, and the four β-hairpins are shown in black. (C) Top view of the β-helix showing the internal side-chain stacking. Residues are shown in stick representation. (D) Stereoview of the 2F_o − F_c electron density map contoured at 1σ of the cross-section of the α^43a β-helix (residues ⁹⁰L–¹⁰⁸H).Ag43 is found in most E. coli pathotypes, including uropathogenic E. coli (UPEC) (10). Many UPEC strains also possess multiple copies of the Ag43-encoding agn43 (or flu) gene (10, 11). Well-characterized functions of Ag43 include cell aggregation and biofilm formation (12–14). Sequence variation within the α⁴³ domain of Ag43 paralogues is associated with different functional properties. For example, the reference UPEC strain CFT073 contains two Ag43 variants (Ag43a and Ag43b) that share 85% sequence identity in the functional α-domain; only Ag43a mediates strong aggregation and biofilm formation and long-term colonization of the mouse urinary tract (10). Ag43 from UPEC strain UTI89 is also associated with the formation of intracellular bacterial communities (IBCs) that resemble biofilms and contribute to chronic urinary tract infection (UTI) (15).The huge diversity of AT proteins in Gram-negative bacteria supports their role in multiple facets of pathogenesis. Although important virulence functions have been ascribed to several different AT proteins, their mechanism of action remains to be elucidated. This is particularly evident in the case of AIDA-I AT proteins, for which our current knowledge of structure–function relationships is either nonexistent or very limited and insufficient to enable a defined mechanistic model for their role in pathogenesis (3, 16). Here we investigate the structure–function relationships of the prototypic AIDA-I–type AT Ag43a from UPEC and show that its functional α^43a domain contains a β-helical structure. Remarkably, unlike other structurally characterized β-helices, α^43a has an L shape, and our work shows a critical interplay between the bent β-helical scaffold and the virulence activity of this protein. Our data also provide unique insight into the mechanism of self-association of Ag43, which may represent a universal mechanism of AT-mediated bacterial aggregation.