Guy Drolet

Expression and neuropeptidergic characterization of estrogen receptors (ER? and ER?) throughout the rat brain: Anatomical evidence of distinct roles of each subtype

The recent cloning of a second estrogen receptor (ER) provided a new tool to investigate and clarify how estrogens are capable of communicating with the brain and influence gene expression and neural function. The purpose of the present study was to define the neuroanatomical organization of each receptor subtype using a side-by-side approach and to characterize the cellular population (s) expressing the ERbeta transcript in the endocrine hypothalamus using immunohistochemistry combined with in situ hybridization. Axonal transport inhibition was accomplished to cause neuropeptide accumulation into the cytoplasm and thus facilitate the detection of all positive luteinizing hormone-releasing hormone (LHRH), corticotropin-releasing factor (CRF), vasopressin (AVP), oxytocin (OT), gastrin-related peptide (GRP), and enkephalin (ENK) neurons. The genes encoding either ERalpha or -beta were expressed in numerous limbic-associated structures, and fine differences were found in terms of intensity and positive signal. Such phenomenon is best represented by the bed nucleus of the stria terminalis (BnST) and preoptic area/anterior hypothalamus, where the expression pattern of both transcripts differed across subnuclei. The novel ER was also found to be expressed quite exclusively in other hypothalamic nuclei, including the supraoptic (SON) and selective compartments (magnocellular and autonomic divisions) of the paraventricular nucleus (PVN). A high percentage of the ERbeta-expressing neurons located in the ventro- and dorsomedial PVN are of OT type; 40% of the OT-ir cells forming the medial magnocellular and ventromedial parvocellular PVN showed a clear hybridization signal for ERbeta mRNA, whereas a lower percentage (15-20%) of OT neurons were positive in the caudal parvocellular PVN and no double-labeled cells were found in the rostral PVN and other regions of the brain with the exception of the SON. Very few AVP-ir neurons expressing ERbeta transcript were found throughout the rat brain, although the medial PVN displayed some scattered double-labeled cells (<5%). Quite interestingly, the large majority of the ERbeta-positive cells in the caudal PVN were colocalized within CRF-ir perikarya. Indeed, more than 60-80% of the CRF-containing cells located in the caudolateral division of the parvocellular PVN exhibited a positive hybridization signal for ERbeta mRNA, whereas very few (<5%) neuroendocrine CRF-ir parvocellular neurons of the medial PVN expressed the gene encoding ERbeta. A small percentage of ERbeta-expressing cells in the dorsocaudal and ventromedial zones of the parvocellular PVN were also ENK positive. The ventral zone of the medial parvocellular PVN also displayed GRP-ir neurons, but no convincing hybridization signal for ERbeta was detected in this neuronal population. Finally, as previously described for the gene encoding the classic ER, LHRH neurons of both intact and colchicine-pretreated animals did not express the novel estrogen receptor. This study shows a differential pattern of expression of both receptors in the brain of intact rats and that ERbeta is expressed at various levels in distinct neuropeptidergic populations, including OT, CRF, and ENK. The influence of estrogen in mediating genomic and neuronal responses may therefore take place within these specific cellular groups in the brains of cycling as well as intact male mammals.

Role of endogenous opioid system in the regulation of the stress response

Numerous studies and reviews support an important contribution of endogenous opioid peptide systems in the mediation, modulation, and regulation of stress responses including endocrine (hypothalamopituitary-adrenal, HPA axis), autonomic nervous system (ANS axis), and behavioral responses. Although several discrepancies exist, the most consistent finding among such studies using different species and stressors is that opioids not only diminish stress-induced neuroendocrine and autonomic responses, but also stimulate these effector systems in the non-stressed state. A distinctive feature of the analgesic action of opioids is the blunting of the distressing, affective component of pain without dulling the sensation itself. Therefore, opioid peptides may diminish the impact of stress by attenuating an array of physiologic responses including emotional and affective states. The widespread distribution of enkephalin (ENK) throughout the limbic system (including the extended amygdala, cingulate cortex, entorhinal cortex, septum, hippocampus, and the hypothalamus) is consistent with a direct role in the modulation the stress responses. The predictability of stressful events reduces the impact of a wide range of stressors and ENK appears to play an important role in this process. Therefore, ENK and its receptors could represent a major modulatory system in the adaptation of an organism to stress, balancing the response that the stressor places on the central stress system with the potentially detrimental effects that a sustained stress may produce. Chronic neurogenic stressors will induce changes in specific components of the stress-induced ENKergic system, including ENK, delta- and mu-opioid receptors. This review presents evidences for adaptive cellular mechanisms underlying the response of the central stress system when assaulted by repeated psychogenic stress, and the involvement of ENK in these processes.

Brief exposure to predator odor and resultant anxiety enhances mesocorticolimbic activity and enkephalin expression in CD-1 mice

The present study assessed alterations in mesolimbic enkephalin (ENK) mRNA levels after predator [2,5‐dihydro‐2,4,5‐trimethylethiazoline (TMT)] and non‐predator (butyric acid) odor encounter and/or light–dark (LD) testing in CD‐1 mice immediately, 24, 48 and 168 h after the initial odor encounter and/or LD testing. The nucleus accumbens, ventral tegmental area, basolateral (BLA), central (CEA) and medial amygdaloid nuclei, prelimbic and infralimbic cortex were assessed for fos‐related antigen (FRA) and/or ENK mRNA as well as neuronal activation of ENK neurons (FRA/ENK). Mice exposed to TMT displayed enhanced freezing and spent less time in the light of the immediate LD test relative to saline‐ or butyric acid‐treated mice. Among mice exposed to TMT, LD anxiety‐like behavior was associated with increased FRA in the prelimbic cortex and accumbal shell and decreased ENK‐positive neurons in the accumbal core. Mice displaying high TMT‐induced LD anxiety exhibited increased ENK‐positive neurons in the BLA, CEA and medial amygdaloid nuclei relative to mice that displayed low anxiety‐like behavior in the LD test after TMT exposure. In the BLA and CEA, ‘high‐anxiety’ mice also displayed increased FRA/ENK after TMT exposure and LD testing. In contrast to neural cell counts, the level of ENK transcript was decreased in the BLA and CEA of ‘high‐anxiety’ mice after TMT exposure and LD testing. These data suggest that increased FRA may regulate stressor‐responsive genes and mediate long‐term behavioral changes. Indeed, increased ENK availability in mesolimbic sites may promote behavioral responses that detract from the aversiveness of the stressor experience.

Neonatal maternal separation and sex-specific plasticity of the hypoxic ventilatory response in awake rat.

We tested the hypothesis that neonatal maternal separation (NMS), a form of stress that affects hypothalamo–pituitary–adrenal axis (HPA) function in adult rats, alters development of the respiratory control system. Pups subjected to NMS were placed in a temperature and humidity controlled incubator 3 h per day for 10 consecutive days (P3 to P12). Control pups were undisturbed. Once they reached adulthood (8–10 weeks old), rats were placed in a plethysmography chamber for measurement of ventilatory and cardiovascular parameters under normoxic and hypoxic conditions. Measurement of c‐fos mRNA expression in the paraventricular nucleus of the hypothalamus (PVH) combined with plasma ACTH and corticosterone levels confirmed that NMS effectively disrupted HPA axis function in males. In males, baseline minute ventilation was not affected by NMS. In contrast, NMS females show a greater resting minute ventilation due to a larger tidal volume. The hypoxic ventilatory response of male NMS rats was 25% greater than controls, owing mainly to an increase in tidal volume response. This augmentation of the hypoxic ventilatory response was sex‐specific also because NMS females show an attenuated minute ventilation increase. Baseline mean arterial blood pressure of male NMS rats was 20% higher than controls. NMS‐related hypertension was not significant in females. The mechanisms underlying sex‐specific disruption of cardio‐respiratory control in NMS rats are unknown but may be a consequence of the neuroendocrine disruption associated with NMS. These data indicate that exposure to a non‐respiratory stress during early life elicits significant plasticity of these homeostatic functions which persists until adulthood.

CRFergic Innervation Of the Paraventricular Nucleus of the Rat Hypothalamus: A Tract‐Tracing Study

It has been reported that corticotropin‐releasing factor (CRF) may regulate its own biosynthesis in the paraventricular nucleus of the hypothalamus (PVH). Whether this CRF autoregulation is mediated by local circuitry or from extra‐PVH CRF neuronal fibers terminating on CRF perikarya within the PVH is unknown. In the present study, we sought to determine the origin(s) of this CRF innervation using retrograde transport of wheat germ‐conjugated‐gold particles (WGA‐apoHRP‐Au) combined with immunohistochemistry for CRF. The rats also received colchicine (100 μg, icv) 5–7 days after tracer injection and were perfused 24 h later. Results of retrograde labeling with pressure injections of WGA‐apoHRP‐Au centered to PVH and subsequent immunohistochemical staining for CRF demonstrated numerous retrogradely labeled CRF neurons in the perifornical hypothalamic nucleus (PeF), the dorsolateral hypothalamic area (DA) (medial and lateral portions) and the dorsomedial nucleus of the hypothalamus (DMH). Smaller groups of CRF‐ir neurons that were retrogradely labeled were found in the bed nuclei of the stria terminalis (BnST), the Barrington’s nucleus (Bar) and the dorsal raphé (DR). These CRFergic pathways to the PVH may represent an anatomical substrate underlying the function of the stress‐integrative PVH neurons in the autonomic, behavioral and neuroendocrine regulation during the stress response, including CRF autoregulation.

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.

Comparisons of Behavioral and Neurochemical Characteristics between WKY, WKHA, and Wistar Rat Strains ☆

WKHA rats constitute a recombinant inbred rat strain derived by phenotypic selection of the progeny of hybrid F2 crosses between SHR and WKY rats. WKHA are normotensive and show some features of hyperactivity and of hyper-reactivity to stress, but their utility as model of attention deficit/hyperactivity disorder (ADHD) has not yet been settled. To address these questions, we performed behavioral and neurochemical evaluations of WKHA, and compared them to both WKY and Wistar (WIS) rats. In locomotor activity tests, the respective scores for each strain were WKY<WKHA<WIS. The relative amplitudes of the inhibition of the startle response by preexposure of the animals to a low-level acoustic cue (prepulse inhibition (PPI) stimulus filtering test) were WKY< WKHA = WIS. In nontreated rats, prepulse inhibition was significantly lower in WKY than in WIS and WKHA rats at low prepulse intensity. Methylphenidate did not decrease locomotor activity in any of the strains, but rather increased locomotion, with the effect being of higher amplitude in WKY. Methylphenidate also impaired prepulse inhibition in a dose-dependent manner. In situ hybridization for NGFI-B, a transcription factor related to the dopaminergic system, revealed that methylphenidate increased the expression of NGFI-B mRNA in the accumbens (core/shell) and striatum only in WKY rats. We conclude that: (1) the behavioral differences between WKY and WKHA can be more readily explained by deficits present in WKY than by unusual characteristics of WKHA, (2) the WKHA does not appear to represent a useful model of ADHD, and (3) the use of WKY rats as the only control for behavioral studies is potentially misleading, because they show a combination of behavioral and neurochemical (dopamine-related) characteristics that make them very different from other common rat strains. Although these characteristics may make them interesting models in their own right, it also follows that the conclusions of any study based on comparisons of one particular strain to just WKY should be interpreted very cautiously.

Increased anxiety-like behaviors in rats experiencing chronic inflammatory pain.

For many patients, chronic pain is often accompanied, and sometimes amplified, by co-morbidities such as anxiety and depression. Although it represents important challenges, the establishment of appropriate preclinical behavioral models contributes to drug development for treating chronic inflammatory pain and associated psychopathologies. In this study, we investigated whether rats experiencing persistent inflammatory pain induced by intraplantar injection of complete Freunds adjuvant (CFA) developed anxiety-like behaviors, and whether clinically used analgesic and anxiolytic drugs were able to reverse CFA-induced anxiety-related phenotypes. These behaviors were evaluated over 28 days in both CFA- and saline-treated groups with a variety of behavioral tests. CFA-induced mechanical allodynia resulted in increased anxiety-like behaviors as evidenced by: (1) a significant decrease in percentage of time spent and number of entries in open arms of the elevated-plus maze (EPM), (2) a decrease in number of central squares visited in the open field (OF), and (3) a reduction in active social interactions in the social interaction test (SI). The number of entries in closed arms in the EPM and the distance traveled in the OF used as indicators of locomotor performance did not differ between treatments. Our results also reveal that in CFA-treated rats, acute administration of morphine (3mg/kg, s.c.) abolished tactile allodynia and anxiety-like behaviors, whereas acute administration of diazepam (1mg/kg, s.c) solely reversed anxiety-like behaviors. Therefore, pharmacological treatment of anxiety-like behaviors induced by chronic inflammatory pain can be objectively evaluated using multiple behavioral tests. Such a model could help identify/validate alternative potential targets that influence pain and cognitive dimensions of anxiety.

Effect of Chronic Psychogenic Stress Exposure on Enkephalin Neuronal Activity and Expression in the Rat Hypothalamic Paraventricular Nucleus

Abstract: This study tested the hypothesis that the activation pattern of enkephalinergic (ENKergic) neurons within the paraventricular nucleus of the hypothalamus (PVH) in response to psychogenic stress is identical whether in response to repeated exposure to the same stress (homotypic; immobilization) or to a novel stress (heterotypic; air jet puff). Rats were assigned to either acute or chronic immobilization stress paradigms (90 min/day for 1 or 10 days, respectively). The chronic group was then subjected to an additional 90‐min session of either heterotypic or homotypic stress. A single 90‐min stress session (immobilization or air jet) increased PVH‐ENK heteronuclear (hn) RNA expression. In chronically stressed rats, exposure to an additional stress session (whether homotypic or heterotypic) continued to stimulate ENK hnRNA expression. Acute immobilization caused a marked increase in the numbers of Fos‐immunoreactive and Fos‐ENK double‐labeled cells in the dorsal and ventral medial parvicellular, and lateral parvicellular subdivisions of the PVH. Chronic immobilization caused an attenuated Fos response (∼ 66%) to subsequent immobilization. In contrast, chronic immobilization did not impair ENKergic neuron activation within the PVH following homotypic or heterotypic stress. These results indicate that within the PVH, chronic psychogenic stress markedly attenuates the Fos response, whereas ENKergic neurons resist habituation, principally within the ventral neuroendocrine portion of the nucleus. This suggests an increase in ENK effect during chronic stress exposure. Homotypic (immobilization) and heterotypic (air jet) psychogenic stressors produce similar responses, including Fos, ENK‐Fos, and ENK hnRNA, within each subdivision of the PVH, suggesting similar processing for painless neurogenic stimuli.

Dynorphin and stress related peptides in rat locus coeruleus: contribution of amygdalar efferents

The interaction between the stress axis and endogenous opioid systems has gained substantial attention, because it is increasingly recognized that stress alters individual sensitivity to opiates. One site at which opiates and stress substrates may interact to have global effects on behavior is within the locus coeruleus (LC). We have previously described interactions of several opioid peptides [e.g., proopiomelanocortin, enkephalin (ENK)] with the stress‐related peptide corticotropin‐releasing factor (CRF) in the LC. To examine further the interactions among dynorphin (DYN), ENK, and CRF in the LC, sections were processed for detection of DYN and CRF or DYN and ENK in rat brain. DYN‐ and CRF‐containing axon terminals overlapped noradrenergic dendrites in this region. Dual immunoelectron microscopy showed coexistence of DYN and CRF; 35% of axon terminals containing DYN were also immunoreactive for CRF. In contrast, few axon terminals contained both DYN and ENK. A potential DYN/CRF afferent is the central nucleus of the amygdala (CeA). Dual in situ hybridization showed that, in CeA neurons, 31% of DYN mRNA‐positive cells colocalized with CRF mRNA, whereas 53% of CRF mRNA‐containing cells colocalized with DYN mRNA. Finally, to determine whether limbic DYN afferents target the LC, the CeA was electrolytically lesioned. Light‐level densitometry of DYN labeling in the LC showed a significant decrease in immunoreactivity on the side of the lesion. Taken together, these data indicate that DYN‐ and CRF‐labeled axon terminals, most likely arising from amygdalar sources, are positioned dually to affect LC function, whereas DYN and ENK function in parallel. J. Comp. Neurol. 508:663–675, 2008.