Differential expression of presynaptic genes in a rat model of postnatal hypoxia: relevance to schizophrenia

Obstetric complications play a role in the pathophysiology of schizophrenia. However, the biological consequences during neurodevelopment until adulthood are unknown. Microarrays have been used for expression profiling in four brain regions of a rat model of neonatal hypoxia as a common factor of obstetric complications. Animals were repeatedly exposed to chronic hypoxia from postnatal (PD) day 4 through day 8 and killed at the age of 150 days. Additional groups of rats were treated with clozapine from PD 120-150. Self-spotted chips containing 340 cDNAs related to the glutamate system (""glutamate chips"") were used. The data show differential (up and down) regulations of numerous genes in frontal (FR), temporal (TE) and parietal cortex (PAR), and in caudate putamen (CPU), but evidently many more genes are upregulated in frontal and temporal cortex, whereas in parietal cortex the majority of genes are downregulated. Because of their primary presynaptic occurrence, five differentially expressed genes (CPX1, NPY, NRXN1, SNAP-25, and STX1A) have been selected for comparisons with clozapine-treated animals by qRT-PCR. Complexin 1 is upregulated in FR and TE cortex but unchanged in PAR by hypoxic treatment. Clozapine downregulates it in FR but upregulates it in PAR cortex. Similarly, syntaxin 1A was upregulated in FR, but downregulated in TE and unchanged in PAR cortex, whereas clozapine downregulated it in FR but upregulated it in PAR cortex. Hence, hypoxia alters gene expression regionally specific, which is in agreement with reports on differentially expressed presynaptic genes in schizophrenia. Chronic clozapine treatment may contribute to normalize synaptic connectivity.

Representational Difference Analysis (RDA) reveals differential expression of conserved as well as novel genes during caste-specific development of the honey bee (Apis mellifera L.) ovary

In highly eusocial insects, such as the honey bee, Apis mellifera, the reproductive bias has become embedded in morphological caste differences. These are most expressively denoted in ovary size, with adult queens having large ovaries consisting of 150-200 ovarioles each, while workers typically have only 1-20 ovarioles per ovary. This morphological differentiation is a result of hormonal signals triggered by the diet change in the third larval instar, which eventually generate caste-specific gene expression patterns. To reveal these we produced differential gene expression libraries by Representational Difference Analysis (RDA) for queen and worker ovaries in a developmental stage when cell death is a prominent feature in the ovarioles of workers, whereas all ovarioles are maintained and extend in length in queens. In the queen library, 48% of the gene set represented homologs of known Drosophila genes, whereas in the worker ovary, the largest set (59%) were ESTs evidencing novel genes, not even computationally predicted in the honey bee genome. Differential expression was confirmed by quantitative RT-PCR for a selected gene set, denoting major differences for two queen and two worker library genes. These included two unpredicted genes located in chromosome 11 (Group11.35 and Group11.31, respectively) possibly representing long non-coding RNAs. Being candidates as modulators of ovary development, their expression and functional analysis should be a focal point for future studies. (C) 2011 Elsevier Ltd. All rights reserved.

Cytoplasmic and nuclear localization of cadherin in honey bee (Apis mellifera L.) gonads

Cadherins are crucial molecules mediating cell-cell interactions between somatic and germline cells in insect and mammalian male and female gonads. We analysed the presence and localization of cadherins in ovaries of honeybee queens and in testes of drones. Transcripts representing two classical cadherins, E-cadherin (shotgun) and N-cadherin, as well as three protocadherins (Starry night, Fat and Fat-like) were detected in gonads of both sexes. Pan-cadherin antibodies, which most probably detect a honeybee N-cadherin, were used in immunolocalization analyses. In the germarium of ovarioles, cadherin-IR (cadherin immunoreactivity) was evidenced as homogeneously distributed in the cytoplasm and as nuclear foci, in both germline and somatic cells. It was also detected in polyfusomes and ring canals. In testiolar tubules, cadherin-IR showed a cytoplasmic and nuclear distributon alike in ovaries. The unexpected nuclear localization and cytoplasmic distribution in ovaries and testes were corroborated by immunogold electron microscopy, which revealed cadherin aggregates associated with electron-dense nuclear structures. With respect to cadherin localization, the honeybee differs from Drosophila, the model for gametogenesis in insects, raising the question as to how differences among solitary and social species may be built into and generated from the general architecture of polytrophic meroistic ovaries. It also indicates the possibility of divergent roles for cadherin in the functional architecture of insect gonads, in general, especially in taxa with high reproductive output.

Differential expression of hypoxia pathway genes in honey bee (Apis mellifera L.) caste development

Diphenism in social bees is essentially contingent on nutrient-induced cellular and systemic physiological responses resulting in divergent gene expression patterns. Analyses of juvenile hormone (JH) titers and functional genomics assays of the insulin-insulin-like signaling (IIS) pathway and its associated branch, target-of-rapamycin (TOR), revealed systemic responses underlying honey bee (Apis mellifera) caste development. Nevertheless, little attention has been paid to cellular metabolic responses. Following up earlier investigations showing major caste differences in oxidative metabolism and mitochondrial physiology, we herein identified honey bee homologs of hypoxia signaling factors, HIF alpha/Sima, HIF beta/Tango and PHD/Fatiga and we investigated their transcript levels throughout critical stages of larval development. Amsima, Amtango and Amfatiga showed correlated transcriptional activity, with two peaks of occurring in both queens and workers, the first one shortly after the last larval molt and the second during the cocoon-spinning phase. Transcript levels for the three genes were consistently higher in workers. As there is no evidence for major microenvironmental differences in oxygen levels within the brood nest area, this appears to be an inherent caste character. Quantitative PCR analyses on worker brain, ovary, and leg imaginal discs showed that these tissues differ in transcript levels. Being a highly conserved pathway and linked to IIS/TOR, the hypoxia gene expression pattern seen in honey bee larvae denotes that the hypoxia pathway has undergone a transformation, at least during larval development, from a response to environmental oxygen concentrations to an endogenous regulatory factor in the diphenic development of honey bee larvae. (C) 2010 Elsevier Ltd. All rights reserved.

Mars is close to venus - Female reproductive proteins are expressed in the fat body and reproductive tract of honey bee (Apis mellifera L.) drones

Vitellogenin (Vg) and lipophorin (Lp) are lipoproteins which play important roles in female reproductive physiology of insects. Both are actively taken up by growing oocytes and especially Vg and its receptor are considered as female-specifically expressed. The finding that the fat body of in honey bee (Apis mellifera) drones synthesizes Vg and is present in hemolymph has long been viewed as a curiosity. The recent paradigm change concerning the role played by Vg in honey bee life history, especially social division of labor, has now led us to investigate whether a physiological constellation similar to that seen in female reproduction may also be represented in the male sex. By means of Western blot analysis we could show that both Vg and Lp are present in the reproductive tract of adult drones, including the accessory (mucus) glands, but apparently are not secreted. Furthermore, we analyzed the transcript levels of the genes encoding these proteins (vg and lp), as well as their putative receptors (Amvgr and Amlpr) in fat body and accessory glands. Whereas lp, vg and Amlpr transcript levels decreased with age in both tissues. Amvgr mRNA levels increased with age in fat body. To our knowledge this is the first report that vitellogenin and its receptor are co-expressed in the reproductive system of a male insect. We interpret these findings as a cross-sexual transfer of a social physiological trait, associated with the rewiring of the juvenile hormone/vitellogenin circuitry that occurred in the female sex of honey bees. (C) 2010 Elsevier Ltd. All rights reserved.

Blockade of NMDA or NO in the dorsal premammillary nucleus attenuates defensive behaviors

The dorsal premammillary nucleus (PMd) is a hypothalamic structure that plays a pivotal role in the processing of predatory threats. Lesions of this nucleus virtually eliminate the expression of defensive responses to predator exposure. However, little is known about the neurotransmitters responsible for these behavioral responses. Since PMd neurons express ionotropic glutamate receptors and exposure to predators have been shown to activate nitric oxide (NO) producing cells in this region, the aim of this study was to verify the involvement of glutamate and NO-mediated neurotransmission in defensive reactions modulated by the PMd. We tested in male Wistar rats the hypothesis that intra-PMd injection of the NMDA receptor antagonist, AP7, or the NO synthase inhibitor, N-propyl-L-arginine (NP), would attenuate behavioral responses induced by cat exposure. Our results showed that both AP7 and NP significantly attenuated the behavioral responses induced by the live cat. These results suggest that the NMDA/NO pathway plays an important role in the behavioral responses mediated by the PMd. (C) 2011 Elsevier Inc. All rights reserved.

The diagonal band of Broca is involved in the pressor pathway activated by noradrenaline microinjected into the periaqueductal gray area of rats

Aims: The dorsal periaqueductal gray area (dPAG) is involved in cardiovascular modulation. Previously, we reported that noradrenaline (NA) microinjection into the dPAG caused a pressor response that was mediated by vasopressin release into the circulation. However, the neuronal pathway that mediates this response is as yet unknown. There is evidence that chemical stimulation of the diagonal band of Broca (dbB) also causes a pressor response mediated by systemic vasopressin release. In the present study, we evaluated the participation of the dbB in the pressor response caused by NA microinjection into the dPAG as well as the existence of neural connections between these areas. Main methods: With the above goal, we verified the effect of the pharmacological ablation of the dbB on the cardiovascular response to NA microinjection into the dPAG of unanesthetized rats. In addition, we microinjected the neuronal tracer biotinylated-dextran-amine (BDA) into the dPAG and looked for efferent projections from the dPAG to the dbB. Key findings: The pharmacologically reversible ablation of the dbB with local microinjection of CoCl(2) significantly reduced the pressor response caused by NA microinjection (15 nmol/50 nL) into the dPAG. In addition, BDA microinjection into the dPAG labeled axons in the dbB, pointing to the existence of direct connections between these areas. Significance: The present results indicate that synapses within the dbB are involved in the pressor pathway activated by NA microinjection into the VAG and direct neural projection from the dPAG to the dbB may constitute the neuroanatomic substrate for this pressor pathway. (C) 2009 Elsevier Inc. All rights reserved.

Mechanisms involved in the pressor response to noradrenaline microinjection into the supraoptic nucleus of unanesthetized rats

We report on the cardiovascular effects of noradrenaline (NA) microinjection into the hypothalamic supraoptic nucleus (SON) as well as the central and peripheral mechanisms involved in their mediation. Microinjections of NA 1, 3, 10, 30 or 45 nmol/100 nL into the SON caused dose-related pressor and bradycardiac response in unanesthetized rats. The response to NA 10 nmol was blocked by SON pretreatment with 15 nmol of the alpha(2)-adrenoceptor antagonist RX821002 and not affected by pretreatment with equimolar dose of the selective alpha(1)-adrenoceptor antagonist WB4101, suggesting that local alpha(2)adrenoceptors mediate these responses. Pretreatment of the SON with the nonselective beta-adrenoceptor antagonist propranolol 15 nmol did not affect the pressor response to NA microinjection of into the SON. Moreover, the microinjection of the 100 nmol of the selective alpha(1)-adrenoceptor agonist methoxamine (MET) into the SON did not cause cardiovascular response while the microinjection of the selective alpha(2)adrenoceptor agonists BHT920 (BHT, 100 nmol) or clonidine (CLO, 5 nmol) caused pressor and bradycardiac responses, similar to that observed after the microinjection of NA. The pressor response to NA was potentiated by intravenous pretreatment with the ganglion blocker pentolinium and was blocked by intravenous pretreatment with the V(1)-vasopressin receptor antagonist dTyr(CH2)5(Me)AVP, suggesting an involvement of circulating vasopressin in this response. In conclusion, our results suggest that pressor responses caused by microinjections of NA into the SON involve activation of local alpha(2)-adrenoceptor receptors and are mediated by vasopressin release into circulation. (c) 2008 Published by Elsevier B.V.

alpha(1)-Adrenoceptors in the lateral septal area modulate food intake behaviour in rats

Background and purpose: Control of food intake is a complex behaviour which involves many interconnected brain structures. The present work assessed if the noradrenergic system in the lateral septum (LS) was involved in the feeding behaviour of rats. Experimental approach: In the first protocol, the food intake of rats was measured. Then non-food-deprived animals received either 100 nL of 21 nmol of noradrenaline or vehicle unilaterally in the LS 10 min after local 10 nmol of WB4101, an alpha(1)-adrenoceptor antagonist, or vehicle. In the second protocol, different doses of WB4101 (1, 10 or 20 nmol in 100 nL) were microinjected bilaterally into the LS of rats, deprived of food for 18 h and food intake was compared to that of satiated animals. Key results: One-sided microinjection of noradrenaline into the LS of normal-fed rats evoked food intake, compared with vehicle-injected control animals, which was significantly reduced by alpha(1)-adrenoceptor antagonism. In a further investigation, food intake was significantly higher in food-deprived animals, compared to satiated controls. Pretreatment of the LS with WB4101 reduced food intake in only food-deprived animals in a dose-related manner, suggesting that the LS noradrenergic system was involved in the control of food intake. Conclusion and implications: Activation by local microinjection of noradrenaline of alpha(1)-adrenoceptors in the LS evoked food intake behaviour in rats. In addition, blockade of the LS alpha(1)-adrenoceptors inhibited food intake in food-deprived animals, suggesting that the LS noradrenergic system modulated food intake behaviour and satiation.

Serotonergic neurotransmission in the dorsal raphe nucleus recruits in situ 5-HT2A/2C receptors to modulate the post-ictal antinociception

The post-ictal immobility syndrome is followed by a significant increase in the nociceptive thresholds in animals and humans. The aim of this study was to assess the involvement of the dorsal raphe nucleus (DRN) in the post-ictal antinociception. The second aim was to study the role of serotonergic intrinsic mechanisms of the DRN in this hypo-algesic phenomenon. Pentylenetetrazole (PTZ), an ionophore GABA-mediated Cl- influx antagonist, was peripherally used to induce tonic-clonic seizures in Wistar rats. The nociceptive threshold was measured by the tail-flick test. Neurochemical lesions of the DRN, performed with microinjection of ibotenic acid (1.0 mu g/0.2 mu L), caused a significant decrease of tonic-clonic seizure-induced antinociception, suggesting the involvement of this nucleus in this antinociceptive Process. Microinjections of methysergide (1.0 and 5.0 mu g/0.2 mu L), a non-selective serotonergic receptor antagonist, into DRN caused a significant decrease in the post-ictal antinociception in seizing animals, compared to controls, in all post-ictal periods Presently studied. These findings were corroborated by microinjections of ketanserin (at 1.0 and 5.0 mu g/0.2 mu L) into DRN. Ketanserin is an antagonist with large affinity for 5-HT2A/2C serotonergic receptors, which, in this Case, Caused a significant decrease in the tail-flick latencies in seizing animals, compared to controls after the first 20 min following tonic-clonic convulsive reactions. These results indicate that serotonergic neurotransmission of the DRN neuronal clusters is involved in the organization of the post-ictal hypo-algesia. The 5-HT2A/2C receptors of DRN neurons seem to be critically involved in the increase of nociceptive thresholds following tonic-clonic seizures. (c) 2008 Elsevier Inc, All rights reserved.

Activation of cannabinoid CB, receptors in the dorsolateral periaqueductal gray induces anxiolytic effects in rats submitted to the Vogel conflict test

There are contradictory results concerning the effects of systemic injections of cannabinoid agonists in anxiety-induced behavioral changes. Direct drug administration into brain structures related to defensive responses could help to clarify the role of cannabinoids in these changes. Activation of cannabinoid CB, receptors in the dorsolateral periaqueductal gray induces anxiolytic-like effects in the elevated plus maze. The aim of this work was to verify if facilitation of endocannabinoid-mediated neurotransmission in this region would also produce anxiolytic-like effects in another model of anxiety, the Vogel conflict test. Male Wistar rats (n = 5-9/group) with cannulae aimed at the dorsolateral periaqueductal gray were water deprived for 24 h and pre-exposed to the apparatus where they were allowed to drink for 3 min. After another 24 h-period of water deprivation, they received the microinjections and, 10 min later, were placed into the experimental box. in this box an electrical shock (0.5 nnA, 2 s) was delivered in the spout of a drinking bottle at every twenty licks. The animals received a first microinjection of vehicle (0.2 mu l) or AM251 (a cannabinoid CB1 receptor antagonist; 100 pmol) followed, 5 min later, by a second microinjection of vehicle, anandamide (an endocannabinoid, 5 pmol), AM404 (an inhibitor of anandamide uptake, 50 pmol) or URB597 (an inhibitor of Fatty Acid Amide Hydrolase, 0.01 or 0.1 nmol). Anandamide, AM404 and URB597 (0.01 nmol) increased the total number of punished licks. These effects were prevented by AM251. The results give further support to the proposal that facilitation of CB1 receptor-mediated endocannabinoid neurotransmission in the dorsolateral periaqueductal gray modulates defensive responses. (C) 2008 Elsevier B.V. All rights reserved.

Cardiovascular responses to microinjection of noradrenaline into the medial amygdaloid nucleus of conscious rats result from alpha(2)-receptor activation and vasopressin release

The medial amygdaloid nucleus (MeA) is involved in the modulation of physiological and behavioral processes, as well as regulation of the autonomic nervous system. Moreover, MeA electrical stimulation evokes cardiovascular responses. Thus, as noradrenergic receptors are present in this structure, the present study tested the effects of local noradrenaline (NA) microinjection into the MeA on cardiovascular responses in conscious rats. Moreover, we describe the types of adrenoceptor involved and the peripheral mechanisms involved in the cardiovascular responses. Increasing doses of NA (3, 9, 27 or 45 nmol/100 nL) microinjected into the MeA of conscious rats caused dose-related pressor and bradycardic responses. The NA cardiovascular effects were abolished by local pretreatment of the MeA with 10 nmol/100 nL of the specific alpha(2)-receptor antagonist RX821002, but were not affected by local pretreatment with 10 nmol/100 nL of the specific alpha(1)-receptor antagonist WB4101. The magnitude of pressor response evoked by NA microinjected into the MeA was potentiated by intravenous pretreatment with the ganglion blocker pentolinium (5 mg/kg), and blocked by intravenous pretreatment with the selective V(1)-vasopressin antagonist dTyr(CH(2))(5)(Me)AVP (50 mu g/kg). In conclusion, our results show that microinjection of NA into the MeA of conscious rats activates local alpha(2)-adrenoceptors, evoking pressor and bradycardic responses, which are mediated by vasopressin release.

Involvement of the lateral habenula in the regulation of generalized anxiety- and panic-related defensive responses in rats

Recently obtained evidence points to the involvement of the lateral habenular nuclei (LHb) in the mediation of coping defensive responses to threatening/stressful stimuli. Nevertheless, the role of this brain area in the regulation of defensive responses that have been associated with specific subtypes of anxiety disorders recognized in clinical settings is presently unknown. To address this question, we investigated the effects of either electrolytic lesions or chemical stimulation of the LHb on the defensive behaviors generated in rats by the elevated T-maze. This experimental model allows the measurement, in a same rat, of two defensive behaviors, inhibitory avoidance and escape, that have been related in terms of psychopathology to generalized anxiety and panic disorders, respectively. Bilateral electrolytic lesions of the LHb (1 mA, 10 s) impaired inhibitory avoidance acquisition and facilitated escape performance. On the other hand, chemical stimulation of the LHb by bilateral microinjection of kainic acid (30-60 pmol/0.2 mu L) had the opposite effect, i.e., facilitated inhibitory avoidance and impaired escape. The present results indicate that the LHb exerts an opposed regulatory control on generalized anxiety- and panic-related defensive responses in rats. (c) 2008 Elsevier Inc. All rights reserved.

Cardiovascular responses to noradrenaline microinjection in the ventrolateral periaqueductal gray of unanesthetized rats

The periaqueductal gray area (PAG) is a mesencephalic area involved in cardiovascular modulation. Noradrenaline (NA), a neurotransmitter involved in central blood pressure control, is present in the rat PAG. We report here on the cardiovascular effects caused by NA microinjection into the ventrolateral PAG (vlPAG) of unanesthetized rats and the peripheral mechanism involved in their mediation. NA microinjection in the vlPAG of unanesthetized rats evoked dose-related pressor and bradycardiac responses. No significant cardiovascular responses were observed in urethane-anesthetized rats. The pressor response was potentiated by pretreatment with the ganglion blocker pentolinium (5 or 10 mg/kg, intravenously). Pretreatment with the vasopressin antagonist dTyr(CH(2))(5) (Me)AVP (50 mu g/kg, intravenously) blocked the pressor response evoked by the NA microinjection into the vlPAG. Additionally, circulating vasopressin content was found to be significantly increased after NA microinjection in the vlPAG. The results suggest that activation of noradrenergic synapses within the vlPAG modulates vasopressin release in unanesthetized rats. (c) 2007 Wiley-Liss, Inc.