Retinoid-Related Receptor (ROR) alpha mRNA Expression Is Altered in the Brain of Male Mice Lacking All Ligand-Binding Thyroid Hormone Receptor (TR) Isoforms

In the vertebrate brain, the thalamus serves as a relay and integration station for diverse neuronal information en route from the periphery to the cortex. Deficiency of TH during development results in severe cerebral abnormalities similar to those seen in the mouse when the retinoic acid receptor (ROR)α gene is disrupted. To investigate the effect of the thyroid hormone recep-tors (TRs) on RORalpha gene expression, we used intact male mice, in which the genes encoding the α and beta TRs have been deleted. In situ hybridization for RORalpha mRNA revealed that this gene is expressed in specific areas of the brain including the thalamus, pons, cerebellum, cortex, and hippocampus. Our quantitative data showed differences in RORalpha mRNA expression in different subthalamic nuclei between wild-type and knock-out mice. For example, the centromedial nucleus of the thalamus, which plays a role in mediating nociceptive and visceral information from the brainstem to the basal ganglia and cortical regions, has less expression of RORalpha mRNA in the knockout mice (-37%) compared to the wild-type controls. Also, in the dorsal geniculate (+72%) and lateral posterior nuclei (+58%) we found more RORalpha mRNA in dKO as compared to dWT animals. Such differences in RORalpha mRNA expression may play a role in the behavioral alterations resulting from congenital hypothyroidism.

Adenosine receptor type 2a is differently modulated by nicotine in dorsal brainstem cells of Wistar Kyoto and spontaneously hypertensive rats

Hypertension can result from neuronal network imbalance in areas of central nervous system that control blood pressure, such as the nucleus tractus solitarius (NTS). There are several neurotransmitters and neuromodulatory substances within the NTS, such as adenosine, which acts on purinoreceptors A(2a) (A(2a)R). The A(2a)R modulates neurotransmission in the NTS where its activation may induce decrease in blood pressure by different mechanisms. Nicotine is a molecule that crosses the hematoencephalic barrier and acts in several areas of central nervous system including the NTS, where it may interact with some neurotransmitter systems and contributes to the development of hypertension in subjects with genetic predisposition to this disease. In this study we first determined A(2a)R binding, protein, and mRNA expression in dorsomedial medulla oblongata of neonate normotensive (WKY) and spontaneously hypertensive rats (SHR). Subsequently, we analyzed the modulatory effects of nicotine on A(2a)R in cell culture in order to evaluate its possible involvement in the development of hypertension. Data showed a decreased A(2a)R binding and increased protein and mRNA expression in tissue sample and culture of dorsal brainstem from SHR compared with those from WKY rats at basal conditions. Moreover, nicotine modulated A(2a)R binding, protein, and mRNA expression in cells from both strains. Interestingly, nicotine decreased A(2a)R binding and increased protein levels, as well as, induced a differential modulation in A(2a)R mRNA expression. Results give us a clue about the mechanisms involved in the modulatory effects of nicotine on A(2a)R as well as hypothesize its possible contribution to the development of hypertension. In conclusion, we demonstrated that A(2a)R of SHR cells which differ from WKY and nicotine differentially modulates A(2a)R in dorsal brainstem cells of SHR and WKY.

Comparative Internal Structure of Dorsal Lips and Radiolar Appendages in Sabellidae (Polychaeta) and Phylogenetic Implications

Fan worms (Sabellidae) possess paired modified prostomial structures at the base of the radiolar crown, dorso-lateral to the mouth, called dorsal lips. The dorsal lips are involved in the sorting of particles collected by the radiolar crown. The range of variation in the morphology of dorsal lips is extensive, and probably this is not only due to adaptations to different environments and feeding preferences but also due to phylogenetic constraints. In this study, we describe and compare the morphology of dorsal lips in a range of sabellid taxa based on histological cross-sections of these structures, and compare our data and terminology with those of previous studies. Dorsal lips are maintained erect in most taxa by a modified radiole fused to them known as dorsal radiolar appendage. We suggest that dorsal radiolar appendages with an internal supporting axis (cellular or acellular) and probably also the ventral lips are synapomorphies of the family. J. Morphol. 272: 302-319, 2011. (C) 2010 Wiley-Liss, Inc.

Dipeptidyl peptidase IV in the hypothalamus and hippocampus of monosodium glutamate obese and food-deprived rats

Proline-specific dipeptidyl peptidases are emerging as a protease family with important roles in the regulation of signaling by peptide hormones related to energy balance. The treatment of neonatal rats with monosodium glutamate (MSG) is known to produce a selective damage on the arcuate nucleus with development of obesity. This study investigates the relationship among dipeptidyl peptidase IV (DPPIV) hydrolyzing activity, CD26 protein, fasting, and MSG model of obesity in 2 areas of the central nervous system. Dipeptidyl peptidase IV and CD26 were, respectively, evaluated by fluorometry, and enzyme-linked immunosorbent assay and reverse transcriptase polymerase chain reaction in soluble (SF) and membrane-bound (MF) fractions from the hypothalamus and hippocampus of MSG-treated and normal rats, submitted or not to food deprivation (FD). Dipeptidyl peptidase IV in both areas was distinguished kinetically as insensitive (DI) and sensitive (DS) to diprotin A. Compared with the controls, MSG and/or FD decreased the activity of DPPIV-DI in the SF and MF from the hypothalamus, as well as the activity of DPPIV-DS in the SF from the hypothalamus and in the MF from the hippocampus. Monosodium glutamate and/or FD increased the activity of DPPIV-DI in the MF from the hippocampus. The monoclonal protein expression of membrane CD26 by enzyme-linked immunosorbent assay decreased in the hypothalamus and increased in the hippocampus of MSG and/or FD relative to the controls. The existence of DPPIV-like activity with different sensitivities to diprotin A and the identity of insensitive with CD26 were demonstrated for the first time in the central nervous system. Data also demonstrated the involvement of DPPIV-DI/CD26 hydrolyzing activity in the energy balance probably through the regulation of neuropeptide Y and beta-endorphin levels in the hypothalamus and hippocampus. (C) 2011 Elsevier Inc. All rights reserved.

Acquisition of Pavlovian Fear Conditioning Using beta-Adrenoceptor Activation of the Dorsal Premammillary Nucleus as an Unconditioned Stimulus to Mimic Live Predator-Threat Exposure

In the present work, we sought to mimic the internal state changes in response to a predator threat by pharmacologically stimulating the brain circuit involved in mediating predator fear responses, and explored whether this stimulation would be a valuable unconditioned stimulus (US) in an olfactory fear conditioning paradigm (OFC). The dorsal premammillary nucleus (PMd) is a key brain structure in the neural processing of anti-predatory defensive behavior and has also been shown to mediate the acquisition and expression of anti-predatory contextual conditioning fear responses. Rats were conditioned by pairing the US, which was an intra-PMd microinjection of isoproterenol (ISO; beta-adrenoceptor agonist), with amyl acetate odor-the conditioned stimulus (CS). ISO (10 and 40 nmol) induced the acquisition of the OFC and the second-order association by activation of beta-1 receptors in the PMd. Furthermore, similar to what had been found for contextual conditioning to a predator threat, atenolol (beta-1 receptor antagonist) in the PMd also impaired the acquisition and expression of OFC promoted by ISO. Considering the strong glutamatergic projections from the PMd to the dorsal periaqueductal gray (dPAG), we tested how the glutamatergic blockade of the dPAG would interfere with the OFC induced by ISO. Accordingly, microinjections of NMDA receptor antagonist (AP5, 6 nmol) into the dPAG were able to block both the acquisition, and partially, the expression of the OFC. In conclusion, we have found that PMd beta-1 adrenergic stimulation is a good model to mimic predatory threat-induced internal state changes, and works as a US able to mobilize the same systems involved in the acquisition and expression of predator-related contextual conditioning. Neuropsychopharmacology (2011) 36, 926-939; doi:10.1038/npp.2010.231; published online 5 January 2011

The non-coding RNA BC1 is down-regulated in the hippocampus of Wistar Audiogenic Rat (WAR) strain after audiogenic kindling

The aim of this study was to identify molecular pathways involved in audiogenic seizures in the epilepsy-prone Wistar Audiogenic Rat (WAR). For this, we used a suppression-subtractive hybridization (SSH) library from the hippocampus of WARs coupled to microarray comparative gene expression analysis, followed by Northern blot validation of individual genes. We discovered that the levels of the non-protein coding (npc) RNA BC1 were significantly reduced in the hippocampus of WARs submitted to repeated audiogenic seizures (audiogenic kindling) when compared to Wistar resistant rats and to both naive WARs and Wistars. By quantitative in situ hybridization, we verified lower levels of BC1 RNA in the GD-hilus and significant signal ratio reduction in the stratum radiatum and stratum pyramidale of hippocampal CA3 subfield of audiogenic kindled animals. Functional results recently obtained in a BC1-/- mouse model and our current data are supportive of a potential disruption in signaling pathways, upstream of BC1, associated with the seizure susceptibility of WARs. (C) 2010 Elsevier B.V. All rights reserved.

Postnatal fluoxetine treatment affects the development of serotonergic neurons in rats

The goal of the present study was to investigate morphological changes in the serotonergic neurons/terminals in the dorsal (DR) and median (MnR) raphe nuclei and on the hippocampal dentate gyrus (DG) in neonatal rats treated from the 1st to the 21st postnatal day with fluoxetine (10 mg/kg sc, daily) or drug vehicle (0 9% saline 1 ml/kg). The results show that postnatal chronic treatment with fluoxetine promoted. (1) a smaller body weight increase during the pre-weaning period; (2) smaller number of 5-HT neurons in the DR, (3) smaller 5-HT neuronal cell bodies (area, perimeter and diameter) in the DR and the MnR and (4) diminished serotonergic terminals in the DG. These data suggest that the development of the serotonergic system was impaired and that early exposure to fluoxetine damaged the morphology of 5-HT neurons in young adult rats While these findings are consistent with other work, more studies are needed to better clarify the effects of postnatal chronic treatment with fluoxetine on the serotonergic system and, consequently, on the functions modulated by serotonin (C) 2010 Elsevier Ireland Ltd All rights reserved

A study of the catecholaminergic inputs to the dorsal premammillary nucleus

The dorsal premammillary nucleus (PMd) is one of the most responsive hypothalamic sites during exposure to a predator or its odor, and to a context previously associated with a predatory threat; and lesions or pharmacological inactivation centered therein severely reduced the anti-predatory defensive responses. Previous studies have shown that beta adrenergic transmission in the PMd seems critical to the expression of fear responses to predatory threats. In the present study, we have investigated the putative sources of catecholaminergic inputs to the PMd. To this end, we have first described the general pattern of catecholaminergic innervation of the PMd by examining the distribution and morphology of the tyrosine hydroxylase (TH) immunoreactive fibers in the nucleus; and next, combining Fluoro Gold (FG) tracing experiments and TH immunostaining, we determined the putative sources of catecholaminergic inputs to the nucleus. Our results revealed that the PMd presents a moderately dense plexus of catecholaminergic fibers that seems to encompass the rostral pole and ventral border of the nucleus. Combining the results of the FG tract-tracing and TH immunostaining, we observed that the locus coeruleus was the sole brain site that contained double FG and TH immunostained cells. In summary, the evidence suggests that the locus coeruleus is seemingly a part of the circuit responding to predatory threats, and, as shown by the present results, is the sole source of catecholaminergic inputs to the PMd, providing noradrenergic inputs to the nucleus, which, by acting via beta adrenoceptor, seems to be critical for the expression of anti-predatory responses. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Increased Expression of GluR2-flip in the Hippocampus of the Wistar Audiogenic Rat Strain After Acute and Kindled Seizures

The Wistar Audiogenic Rat (WAR) is an epileptic-prone strain developed by genetic selection from a Wistar progenitor based on the pattern of behavioral response to sound stimulation. Chronic acoustic stimulation protocols of WARs (audiogenic kindling) generate limbic epileptogenesis, confirmed by ictal semiology, amygdale, and hippocampal EEG, accompanied by hippocampal and amygdala cell loss, as well as neurogenesis in the dentate gyrus (DG). In an effort to identify genes involved in molecular mechanisms underlying epileptic process, we used suppression-subtractive hybridization to construct normalized cDNA library enriched for transcripts expressed in the hippocampus of WARs. The most represented gene among the 133 clones sequenced was the ionotropic glutamate receptor subunit II (GluR2), a member of the a-amino-3-hydroxy-5-methyl-4-isoxazoleopropionic acid (AMPA) receptor. Although semiquantitative RT-PCR analysis shows that the hippocampal levels of the GluR2 subunits do not differ between naive WARs and their Wistar counterparts, we observed that the expression of the transcript encoding the splice-variant GluR2-flip is increased in the hippocampus of WARs submitted to both acute and kindled audiogenic seizures. Moreover, using in situ hybridization, we verified upregulation of GluR2-flip mainly in the CA1 region, among the hippocampal subfields of audiogenic kindled WARs. Our findings on differential upregulation of GluR2-flip isoform in the hippocampus of WARs displaying audiogenic seizures is original and agree with and extend previous immunohistochemical for GluR2 data obtained in the Chinese P77PMC audiogenic rat strain, reinforcing the association of limbic AMPA alterations with epileptic seizures. (C) 2009 Wiley-Liss, Inc.

Sensing danger through the olfactory system: The role of the hypothalamic dorsal premammillary nucleus

The dorsal premammillary nucleus (PMd) has a critical role on the expression of defensive responses to predator odor. Anatomical evidence suggests that the PMd should also modulate memory processing through a projecting branch to the anterior thalamus. By using a pharmacological blockade of the PMd with the NMDA-receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5), we were able to confirm its role in the expression of unconditioned defensive responses, and further revealed that the nucleus is also involved in influencing associative mechanisms linking predatory threats to the related context. We have also tested whether olfactory fear conditioning, using coffee odor as CS, would be useful to model predator odor. Similar to cat odor, shock-paired coffee odor produced robust defensive behavior during exposure to the odor and to the associated context. Shock-paired coffee odor also up-regulated Fos expression in the PMd, and, as with cat odor, we showed that this nucleus is involved in the conditioned defensive responses to the shock-paired coffee odor and the contextual responses to the associated environment. (C) 2008 Elsevier Ltd. All rights reserved.

Expression of alpha-synuclein is increased in the hippocampus of rats with high levels of innate anxiety

A genomic region neighboring the alpha-synuclein gene, on rat chromosome 4, has been associated with anxiety- and alcohol-related behaviors in different rat strains. In this study, we have investigated potential molecular and physiological links between alpha-synuclein and the behavioral differences observed between Lewis (LEW) and Spontaneously Hypertensive (SHR) inbred rats, a genetic model of anxiety. As expected, LEW rats appeared more fearful than SHR rats in three anxiety models: open field, elevated plus maze and light/dark box. Moreover, LEW rats displayed a higher preference for alcohol and consumed higher quantities of alcohol than SHR rats. alpha-Synuclein mRNA and protein concentrations were higher in the hippocampus, but not the hypothalamus of LEW rats. This result inversely correlated with differences in dopamine turnover in the hippocampus of LEW and SHR rats, supporting the hypothesis that alpha-synuclein is important in the downregulation of dopamine neurotransmission. A novel single nucleotide polymorphism was identified in the 30-untranslated region (3`-UTR) of the alpha-synuclein cDNA between these two rat strains. Plasmid constructs based on the LEW 3`-UTR sequence displayed increased expression of a reporter gene in transiently transfected PC12 cells, in accordance with in-vivo findings, suggesting that this nucleotide exchange might participate in the differential expression of alpha-synuclein between LEW and SHR rats. These results are consistent with a novel role for alpha-synuclein in modulating rat anxiety- like behaviors, possibly through dopaminergic mechanisms. Since the behavioral and genetic differences between these two strains are the product of independent evolutionary histories, the possibility that polymorphisms in the alpha-synuclein gene may be associated with vulnerability to anxiety- related disorders in humans requires further investigation. Molecular Psychiatry (2009) 14, 894-905; doi: 10.1038/mp.2008.43; published online 22 April 2008

Hypothalamic sites responding to predator threats - the role of the dorsal premammillary nucleus in unconditioned and conditioned antipredatory defensive behavior

In this study we provide a comprehensive analysis of the hypothalamic activation pattern during exposure to a live predator or an environment previously associated with a predator. Our results support the view that hypothalamic processing of the actual and the contextual predatory threats share the same circuit, in which the dorsal premammillary nucleus (PMd) plays a pivotal role in amplifying this processing. To further understand the role of the PMd in the circuit organizing antipredatory defensive behaviors, we studied rats with cytotoxic PMd lesions during cat exposure and examined the pattern of behavioral responses as well as how PMd lesions affect the neuronal activation of the systems engaged in predator detection, in contextual memory formation and in defensive behavioral responses. Next, we investigated how pharmacological blockade of the PMd interferes with the conditioned behavioral responses to a context previously associated with a predator, and how this blockade affects the activation pattern of periaqueductal gray (PAG) sites likely to organize the conditioned behavioral responses to the predatory context. Behavioral observations indicate that the PMd interferes with both unconditioned and conditioned antipredatory defensive behavior. Moreover, we have shown that the PMd influences the activation of its major projecting targets, i.e. the ventral part of the anteromedial thalamic nucleus which is likely to influence mnemonic processing, and PAG sites involved in the expression of antipredatory unconditioned and conditioned behavioral responses. Of particular relevance, this work provides evidence to elucidate the basic organization of the neural circuits integrating unconditioned and contextual conditioned responses to predatory threats.

The indirect amygdala-dorsal striatum pathway mediates conditioned freezing: Insights on emotional memory networks

The dorsal striatum (DS) is involved in various forms of learning and memory such as procedural learning, habit learning, reward-association and emotional learning. We have previously reported that bilateral DS lesions disrupt tone fear conditioning (TFC), but not contextual fear conditioning (CFC) [Ferreira TL, Moreira KM, Ikeda DC, Bueno OFA, Oliveira MGM (2003) Effects of dorsal striatum lesions in tone fear conditioning and contextual fear conditioning. Brain Res 987:17-24]. To further elucidate the participation of DS in emotional learning, in the present study, we investigated the effects of bilateral pretest (postraining) electrolytic DS lesions on TFC. Given the well-acknowledged role of the amygdala in emotional learning, we also examined a possible cooperation between DS and the amygdala in TFC, by using asymmetrical electrolytic lesions, consisting of a unilateral lesion of the central amygdaloid nucleus (CeA) combined to a contralateral DS lesion. The results show that pre-test bilateral DS lesions disrupt TFC responses, suggesting that DS plays a role in the expression of TFC. More importantly, rats with asymmetrical pre-training lesions were impaired in TFC, but not in CFC tasks. This result was confirmed with muscimol asymmetrical microinjections in DS and CeA, which reversibly inactivate these structures. On the other hand, similar pretest lesions as well as unilateral electrolytic lesions of CeA and DS in the same hemisphere did not affect TFC. Possible anatomical substrates underlying the observed effects are proposed. Overall, the present results underscore that other routes, aside from the well-established CeA projections to the periaqueductal gray, may contribute to the acquisition/consolidation of the freezing response associated to a TFC task. It is suggested that CeA may presumably influence DS processing via a synaptic relay on dopaminergic neurons of the substantia nigra compacta and retrorubral nucleus. The present observations are also in line with other studies showing that TFC and CFC responses are mediated by different anatomical networks. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.