Activation of signaling pathways downstream of the brain-derived neurotrophic factor receptor, TrkB, in the rat brain by vagal nerve stimulation and antidepressant drugs.

Vagal nerve stimulation (VNS) has been approved for treatment resistant depression (TRD) by the Food and Drug Administration (FDA) since 2005. However, the cellular and molecular targets responsible for its effects are still not characterized. Previously, chronic administration of VNS to rats was found to phosphorylate tyrosine 515 on TrkB, the neurotrophin receptor, whereas traditional antidepressants did not do this. In the present study, Western blot analysis was used to characterize activation due to phosphorylation in the hippocampus of down-stream pathways linked to specific key tyrosine residues on TrkB (namely Y816 and Y515) after either acute or chronic administration of VNS and traditional antidepressant drugs. Chronic administration of VNS caused phosphorylation of effectors linked to Y 515; namely Akt, ERK and p70S6 kinase, but this was not produced by either desipramine or sertraline. All the treatments, when given chronically, caused phosphorylation of the transcription factor, CREB. Acute administration of all the treatments also caused phosphorylation of PLCγ1 but this was not maintained with chronic treatment. Further research is required to determine what role, if any, activation of down-stream targets of Y515 plays in the behavioural effects of VNS.

Intracerebroventricular losartan infusion modulates angiotensin II type 1 receptor expression in the subfornical organ and drinking behaviour in bile-duct-ligated rats.

Bile duct ligation (BDL) causes congestive liver failure that initiates haemodynamic changes, including peripheral vasodilatation and generalized oedema. Peripheral vasodilatation is hypothesized to activate compensatory mechanisms, including increased drinking behaviour and neurohumoral activation. This study tested the hypothesis that changes in the expression of angiotensin II type 1 receptor (AT(1)R) mRNA and protein in the lamina terminalis are associated with BDL-induced hyposmolality in the rat. All rats received either BDL or sham-ligation surgery. The rats were housed in metabolic chambers for measurement of fluid and food intake and urine output. Expression of AT(1)R in the lamina terminalis was assessed by Western blot and quantitative real-time PCR (RT-qPCR). Average baseline water intake increased significantly in BDL rats compared with sham-operated rats, and upregulation of AT(1)R protein and AT(1a)R mRNA were observed in the subfornical organ of BDL rats. Separate groups of BDL and sham-ligated rats were instrumented with minipumps filled with either losartan (2.0 µg µl(-1)) or 0.9% saline for chronic intracerebroventricular or chronic subcutaneous infusion. Chronic intracerebroventricular losartan infusion attenuated the increased drinking behaviour and prevented the increased abundance of AT(1)R protein in the subfornical organ in BDL rats. Chronic subcutaneous infusion did not affect water intake or AT(1)R abundance in the subfornical organ. The data presented here indicate a possible role of increased central AT(1)R expression in the regulation of drinking behaviour during congestive cirrhosis.

Mechanisms associated with the antidepressant-like effects of L-655,708.

Previous research has demonstrated that selective modulation of hippocampal transmission by systemic administration of an α5-GABAA receptor negative allosteric modulator, L-655,708, reproduces the sustained antidepressant-like (AD-like) effect of R,S-ketamine in the absence of any psychotomimetic or abuse-related effects. Pharmacological, electrophysiological (whole-cell patch clamp), and behavioral approaches were used to examine the mechanisms by which L-655,708 produces plasticity within the hippocampus that accounts for its sustained AD-like effect in rats. Inhibitors of either transcription or translation prevented the sustained AD-like effect of L-655,708. Unlike R,S-ketamine, L-655,708 did not cause an increase in the phosphorylation of the receptor for BDNF, TrkB, in the ventral hippocampus (vHipp) 30 or 60 min after its administration nor did administration of the TrkB inhibitor, K252a, directly into the vHipp, block the sustained AD-like effect of L-655,708. Similar to previous results with R,S-ketamine, administration of L-655,709 increased levels of GluA1 in the mPFC and, blockade of such receptors by direct administration of NBQX into the mPFC blocked the sustained AD-like effect of L-655,708. Patch-clamp recordings of ventral CA1 pyramidal cells 24 h after a single systemic administration of L-655,708 revealed a significant increase in input resistance, which resulted in an approximately two-fold increase in action potential frequency. These experiments indicate that the sustained AD-like effects of L-655,708 require protein synthesis and plasticity of GluA1 glutamate receptors in the mPFC. The drug also caused changes in GABAA receptor gating properties in the vHipp with resultant changes in ventral CA1 that indirectly increases neuronal excitability. Such effects likely contribute to its sustained AD-like activity.

Cloning and expression of calglandulin, a new EF-hand protein from the venom glands of Bothrops insularis snake in E. coli.

The EF-hand protein family is comprised of many proteins with conserved Ca(2+)-binding motifs with important biological roles in intracellular communication. During the generation of Expressed Sequence Tags (ESTs) from the venom glands of the Viperidae snake Bothrops insularis, we identified a cDNA coding for a putative Ca(2+) binding protein with four EF-hand motifs, named here calglandulin. The deduced amino acid sequence displayed the greatest sequence similarity with calmodulin (59%), followed by troponin-C (52%). The encoded polypeptide was first expressed in Escherichia coli as a 6XHis-tagged fusion protein. The expressed protein was purified by Ni(2+)-charged affinity chromatography and circular dichroism (CD) spectroscopy confirmed the prevalence of alpha-helix as observed in calmodulin/calmodulin-like proteins. A polyclonal antiserum was generated in mice using this recombinant calglandulin. To investigate the tissue-specific biological occurrence of this protein, this antiserum was used in Western blot experiments, which revealed an immunoreactive band in samples of venom gland extracts from different snakes, but not in the crude venom or in brain, heart and other tissues. This exclusive occurrence suggests a specialized function of calglandulin in snake venom glands.

Cell junctions in the germinal epithelium may play an important role in spermatogenesis of the catfish P. fasciatum (Pisces, Siluriformes).

We identified adhesive junctions and gap junctions between Sertoli cells, between Sertoli and germ cells and between germ cells in the testis of P. fasciatum, a catfish of commercial relevance. To investigate the role of these junctions in spermatogenesis, as well as the molecular composition of the junctions, we performed an immunohistochemistry light microscopy as well as an immunogold labelling electron microscopy study with antibodies to adhesive and gap junctions proteins. Testes that were at different stages of spermatogenesis were used. Based on our morphological studies we speculate that Sertoli-germ and germ-germ cell adhesive junctions are important for maintaining the three-dimensional structure of the germinal cysts and an organized arrangement of the germ cells inside the cysts. Connexin 32 was identified in the germ cells and in the cysts walls. Our observations also suggest that Sertoli-germ and germ-germ cells gap junctions may be involved in the mechanism of synchronous development of germ cells.

Therapeutic modalities for treatment resistant depression: focus on vagal nerve stimulation and ketamine.

Treatment resistant depression (TRD) is a global health concern affecting a large proportion of depressed patients who then require novel therapeutic options. One such treatment option that has received some attention in the past several years is vagal nerve stimulation (VNS). The present review briefly describes the relevance of this treatment in the light of other existing pharmacological and non-pharmacological options. It then summarizes clinical findings with respect to the efficacy of VNS. The anatomical rationale for its efficacy and other potential mechanisms of its antidepressant effects as compared to those employed by classical antidepressant drugs are discussed. VNS has been approved in some countries and has been used for patients with TRD for quite some time. A newer, fast-acting, non-invasive pharmacological option called ketamine is currently in the limelight with reference to TRD. This drug is currently in the investigational phase but shows promise. The clinical and preclinical findings related to ketamine have also been summarized and compared with those for VNS. The role of neurotrophin factors, specifically brain derived neurotrophic factor and its receptor, in the beneficial effects of both VNS and ketamine have been highlighted. It can be concluded that both these therapeutic modalities, while effective, need further research that can reveal specific targets for intervention by novel drugs and address concerns related to side-effects, especially those seen with ketamine.

Vagal nerve stimulation rapidly activates brain-derived neurotrophic factor receptor TrkB in rat brain.

BACKGROUND: Vagal nerve stimulation (VNS) has been approved for treatment-resistant depression. Many antidepressants increase expression of brain-derived neurotrophic factor (BDNF) in brain or activate, via phosphorylation, its receptor, TrkB. There have been no studies yet of whether VNS would also cause phosphorylation of TrkB. METHODS: Western blot analysis was used to evaluate the phosphorylation status of TrkB in the hippocampus of rats administered VNS either acutely or chronically. Acute effects of VNS were compared with those caused by fluoxetine or desipramine (DMI) whereas its chronic effects were compared with those of sertraline or DMI. RESULTS: All treatments, given either acutely or chronically, significantly elevated phosphorylation of tyrosines 705 and 816 on TrkB in the hippocampus. However, only VNS increased the phosphorylation of tyrosine 515, with both acute and chronic administration causing this effect. Pretreatment with K252a, a nonspecific tyrosine kinase inhibitor, blocked the phosphorylation caused by acute VNS at all three tyrosines. Downstream effectors of Y515, namely Akt and ERK, were also phosphorylated after acute treatment with VNS, whereas DMI did not cause this effect. CONCLUSION: VNS rapidly activates TrkB phosphorylation and this effect persists over time. VNS-induced phosphorylation of tyrosine 515 is distinct from the effect of standard antidepressant drugs.

Altered central TRPV4 expression and lipid raft association related to inappropriate vasopressin secretion in cirrhotic rats.

Inappropriate vasopressin (AVP) release causes dilutional hyponatremia in many pathophysiological states such as cirrhosis. The central molecular mechanisms that mediate inappropriate AVP release are unknown. We tested the hypothesis that changes in the expression or trafficking of TRPV4 in the central nervous system may contribute to inappropriate AVP release in the bile duct ligation (BDL) model of cirrhosis in the rat. Four weeks after surgery, BDL rats demonstrated significantly increased plasma vasopressin and plasma renin activity (PRA), hypervolemia, and decreased plasma osmolality. These effects were blocked by providing BDL rats with 2% saline to drink for 15 days. TRPV4 protein expression was significantly increased in brain punches from BDL rats containing the supraoptic nucleus (SON) of the hypothalamus (100% +/- 11 to 157% +/- 4.8), and this effect was blocked in BDL rats given saline. Immunohistochemistry demonstrated a significant increase in TRPV4-positive cells and the percentage of AVP neurons that also were TRPV4-positive in the SON of BDL rats. In the hypothalamus of BDL rats, TRPV4 lipid raft association increased compared with sham (from 100% +/- 2.1 to 326.1% +/- 16). This effect was significantly attenuated in BDL rats given 2% saline to drink (174% +/- 11). In the brain stem, TRPV4 lipid raft association was reduced by BDL and inversely related to plasma AVP and PRA. We speculate that changes in TRPV4 expression and compartmentalization within lipid rafts could contribute to a feed-forward mechanism related to AVP release in cirrhosis.