Jean-François Poulin

Enkephalinergic Afferents of the Centromedial Amygdala in the Rat

The connectivity of the amygdaloid complex has been extensively explored with both anterograde and retrograde tracers. Even though the afferents of the centromedial amygdala [comprising the central (CEA) and medial (MEA) amygdaloid nuclei] are well established, relatively little is known about the neuropeptide phenotype of these connections. In this study, we first examined the distribution of μ‐opioid receptor (MOR) and δ‐opioid receptor (DOR) in the amygdala via in situ hybridization and immunohistochemistry. We then investigated the distribution of Met‐enkephalin (ENK) and Leu‐ENK fibers with immunohistochemistry and examined the distribution of preproenkephalin mRNA in the amygdala by using in situ hybridization. Finally, we examined the ENK projections to the CEA and MEA by using stereotaxic injections of the retrograde tracer cholera toxin subunit B or fluorogold revealed by immunohistochemistry combined with in situ hybridization to identify ENKergic neurons. Our results indicate that the centromedial amygdala receives ENK afferents, as indicated by the presence of MOR, DOR, and ENK fibers in the CEA and MEA, originating primarily from the bed nucleus of the stria terminalis (BST) and from other amygdaloid nuclei. The posterior BST, the basomedial nucleus (BMA), and the cortical nucleus of the amygdala (COA) were found to be the major ENK afferents of the MEA, whereas the anterolateral BST, the COA, the MEA, and the BMA provided the main ENKergic innervation of the CEA. In addition, we found that the ventromedial nucleus of the hypothalamus and the pontine parabrachial nucleus provide a moderate ENK input to the CEA and MEA. The functional implications of these connections in stress, anxiety, and nociception are discussed. J. Comp. Neurol. 496:859–876, 2006.

β-Lactoglobulin tablets as a suitable vehicle for protection and intestinal delivery of probiotic bacteria

The use of succinylated β-lactoglobulin as a novel functional tablet excipient for the protection of probiotic bacteria against the adverse gastric conditions and their delivery in the intestine was studied. Tablets were produced by direct compression of a dry mixture of Bifidobacterium longum HA-135 and the tested excipient. The results showed that tablets made of native β-lg did not ensure cell survival while grafting carboxylic acid groups on the protein revealed to be an innovative method to create a gastroresistant matrix that could allow the survival of up to 10(8)CFU and 10(7)CFU after 1h and 2h gastric incubation, respectively. When compared to other polymers, succinylated β-lg promoted the best survival both upon compression and after simulated gastric passage. The proportion of succinylated β-lg in the formulation could be lowered to 60% without modifying the protective ability of the matrix. Additionally, the tablets proved to be stable over a period of 3 months when refrigerated. Succinylated β-lg tablets are an interesting vehicle for the protection of acid-sensitive bacteria during transit in the upper gastro-intestinal tract.

Impact of feed solution flow rate on Peptide fractionation by electrodialysis with ultrafiltration membrane.

Recently, processes combining an electrical field as a driving force to porous membranes have been developed for the separation of protein or peptide mixtures to obtain more purified products with higher functionality or nutritional value. The objective of this work was to evaluate the influence of the flow rate on the productivity and selectivity as well as on the electrodialytic parameters of electrodialysis with an ultrafiltration membrane (EDUF) during the fractionation of peptides from a beta-lactoglobulin tryptic hydrolysate. It appeared that the feed solution flow rate had no impact on the yield of the process but induced changes in the selectivity. In fact, increases in the flow rate decreased the migration of the peptides with limited electrophoretic mobility.

Enkephalin knockdown in the basolateral amygdala reproduces vulnerable anxiety-like responses to chronic unpredictable stress.

The endogenous enkephalins (ENKs) are potential candidates participating in the naturally occurring variations in coping styles and determining the individual capacities for adaptation during chronic stress exposure. Here we demonstrate that there is a large variance in individual behavioral, as well as in physiological outcomes, in a population of Sprague–Dawley rats subjected to 3 weeks of chronic unpredictable stress (CUS). Separation of resilient and vulnerable subpopulations reveals specific long-term neuroadaptation in the ENKergic brain circuits. ENK mRNA expression was greatly reduced in the posterior basolateral nucleus of amygdala (BLAp) in vulnerable individuals. In contrast, ENK mRNA levels were similar in resilient and control (unstressed) individuals. Another group of rats were used for lentiviral-mediated knockdown of ENK to assess whether a decrease of ENK expression in the BLAp reproduces the behavioral disturbances found in vulnerable individuals. ENK knockdown specifically located in the BLAp was sufficient to increase anxiety in the behavioral tests, such as social interaction and elevated plus maze when compared with control individuals. These results show that specific neuroadaptation mediated by the ENKergic neurotransmission in the BLAp is a key regulator of resilience, whereas a decrease of the ENK in the BLAp is a maladaptation mechanism, which mediates the behavioral dichotomy observed between vulnerable and resilient following 3 weeks of CUS.

Enkephalin knockdown in the central amygdala nucleus reduces unconditioned fear and anxiety

The endogenous opioid enkephalins (ENK) are highly expressed in the central nucleus of the amygdaloid complex (CeA) where several lines of evidence point to a potential role in the modulation of fear and anxiety. In this study, we aimed to assess the role of CeA ENK using local injections of a lentiviral vector expressing a short hairpin RNA (shRNA) targeting ENK in Sprague–Dawley rats. We injected this vector in the CeA and a 56% downregulation of ENK mRNA was observed in animals when compared with scrambled shRNA animals. Anxiety‐like behaviors were also assessed using the elevated plus maze and social interaction test. There was an increase in exploration of open arms of the elevated plus maze in ENK knockdown animals compared with controls, but no change in social interaction. In addition, we used the contextual fear conditioning procedure to assess fear expression and learning in these animals. There was a reduction in freezing induced by acute shocks during the training procedure. Interestingly, associative learning was not affected, and ENK knockdown animals displayed an equivalent freezing when re‐exposed to the conditioning chamber 48 h later. These results contrast with knockout mice studies, which ascribed anxiolytic properties to ENK, and they demonstrate the need for a thorough understanding and characterization of neuroanatomically distinct ENK pathways.

Enkephalin downregulation in the nucleus accumbens underlies chronic stress-induced anhedonia

Abstract Restraint and immobilization have been extensively used to study habituation of the neuroendocrine response to a repeated stressor, but behavioral consequences of this stress regimen remain largely uncharacterized. In this study, we used sucrose preference and the elevated-plus maze to probe behavioral alterations resulting from 14 days of restraint in rats. We observed a decrease in sucrose preference in stressed animals, particularly in a subgroup of individuals, but no alteration in anxiety behaviors (as measured in the elevated-plus maze) four days following the last restraint. In these low-sucrose preference animals, we observed a downregulation of the expression of preproenkephalin mRNA in the nucleus accumbens. Furthermore, we observed a strong correlation between enkephalin expression and sucrose preference in the shell part of the nucleus accumbens, with a lower level of enkephalin expression being associated with lower sucrose preference. Interestingly, quantification of the corticosterone response revealed a delayed habituation to restraint in the low-sucrose preference population, which suggests that vulnerability to stress-induced deficits might be associated with prolonged exposure to glucocorticoids. The induction of ΔFosB is also reduced in the nucleus accumbens shell of the low-sucrose preference population and this transcription factor is expressed in enkephalin neurons. Taken together, these results suggest that a ΔFosB-mediated downregulation of enkephalin in the nucleus accumbens might underlie the susceptibility to chronic stress. Further experiments will be needed to determine causality between these two phenomena.

Effect of Conditioning Ultrafiltration Membranes on their Performances in Electrodialysis with Ultrafiltration Membrane

Abstract A recent alternative method based on ultrafiltration membrane stacked in an electrodialysis cell was recently used for the separation of bio‐active high added value charged molecules, such as peptides and polyphenols. However, the ultrafiltration membranes which are uncharged membranes present lower electrical conductivity, in comparison with conventional ion‐exchange membranes. The purpose of this study was to evaluate the effect of conditioning ultrafiltration membranes of different molecular weight cut‐off (MWCO) (10, 20, 50, and 100 kDa) in solution with different ionic strength (distilled water and 0.1 M NaCl(aq)) on their electrodialytic properties. It appeared that the conditioning solution could have a major impact on the electrical conductivity value of an ultrafiltration membrane and that the final conductivity value after soaking increased with an increase in molecular weight cut‐off. However, the soaking period and solution had no effect on membrane thickness. Furthermore, the electrical conductivity of the membrane was increased after an electrodialysis with ultrafiltration membranes process of a salt solution.