Ana Isabel Santos

Microglial activation, increased TNF and SERT expression in the prefrontal cortex define stress-altered behaviour in mice susceptible to anhedonia

A chronic stress paradigm comprising exposure to predation, tail suspension and restraint induces a depressive syndrome in C57BL/6J mice that occurs in some, but not all, animals. Here, we sought to extend our behavioural studies to investigate how susceptibility (sucrose preference<65%) or resilience (sucrose preference>65%) to stress-induced anhedonia affects the 5HT system and the expression of inflammation-related genes. All chronically stressed animals, displayed increased level of anxiety, but susceptible mice exhibited an increased propensity to float in the forced swim test and demonstrate hyperactivity under stressful lighting conditions. These changes were not present in resilient or acutely stressed animals. Compared to resilient animals, susceptible mice showed elevated expression of tumour necrosis factor alpha (TNF) and the 5-HT transporter (SERT) in the pre-frontal area. Enhanced expression of 5HT(2A) and COX-1 in the pre-frontal area was observed in all stressed animals. In turn, indoleamine-2,3-dioxygenase (IDO) was significantly unregulated in the raphe of susceptible animals. At the cellular level, increased numbers of Iba-1-positive microglial cells were also present in the prefrontal area of susceptible animals compared to resilient animals. Consequently, the susceptible animals display a unique molecular profile when compared to resilient, but anxious, animals. Unexpectedly, this altered profile provides a rationale for exploring anti-inflammatory, and possibly, TNF-targeted therapy for major depression.

Pregnancy or stress decrease complexity of CA3 pyramidal neurons in the hippocampus of adult female rats.

Pregnancy is a time of distinct neural, physiological and behavioral plasticity in the female. It is also a time when a growing number of women are vulnerable to stress and experience stress-related diseases, such as depression and anxiety. However, the impact of stress during gestation on the neurobiology of the mother has yet to be determined, particularly with regard to changes in the hippocampus; a brain area that plays an important role in stress-related diseases. Therefore, the aim of the present study was to understand how stress and reproductive state may alter dendritic morphology of CA1 and CA3 pyramidal neurons in the hippocampus. To do this, adult age-matched pregnant and virgin female Wistar rats were divided into two conditions: (1) control and (2) stress. Females in the stress condition were restrained for 1h/day for the last 2 weeks of gestation and at matched time-points in virgin females. Females were sacrificed the day after the last restraint session and brains were processed for Golgi impregnation. Dendritic length and number of branch points were quantified for apical and basal regions of CA1 and CA3 pyramidal neurons. Results show that regardless of reproductive state, stressed females had significantly shorter apical dendrites and fewer apical branch points in CA3 pyramidal cells. In addition, pregnant females, regardless of stress exposure, had less complex CA3 pyramidal neurons, as measured by Sholl analysis. No differences between conditions were seen in morphology of CA1 pyramidal neurons. This work shows that both repeated restraint stress and pregnancy affect dendritic morphology by decreasing complexity of CA3, but not CA1, neurons in the hippocampus.

Potassium currents in acutely isolated maturing rat hippocampal CA1 neurones

Whole-cell voltage clamp techniques were used to characterize the postnatal development of current amplitude and inactivation and activation kinetics of two potassium currents in acutely isolated CA1 cells from rats P4 to P52: the A-current (IA) and a slow-rising, slow inactivating current (IK). In the course of maturation, changes in the relative proportion of IA and IK currents were observed, the latter becoming a dominant current in older cells. The half-maximal point (Vh) of steady-state inactivation and activation of IA and IK shifted in the course of the first and second postnatal weeks. The shifts were hyperpolarizing in the case of IK, whereas IA shifted to less negative values. The shifts in steady-state inactivation Vh were accompanied by a change in the slope factor (Vs), which is an indication of a modification in the voltage sensitivity of the steady-state inactivation. The kinetics of IK evolve after birth in a fashion that matches the changes in action potential parameters previously reported.

Afterpotential characteristics and firing patterns in maturing rat hippocampal CA1 neurones in in vitro slices.

The postnatal evolution of depolarizing after-potentials (DAPs) and after-hyperpolarizations (AHPs) was studied in rat CA1 hippocampal neurones (5-68 days of age) using in vitro slices. Results were pooled into 4 age groups: P5-9, P10-16, P17-24 and P26-68. In P5-9 cells, DAPs were seen as passive signals, with a time constant similar to the time constant of the membrane. The evolution of the DAP was characterized by a decrease in amplitude, an increase in duration and a change in contour. In P10-16 and P17-24 cells, the DAPs often had a plateau or a hump-like shape which increased the probability of firing and the occurrence of spike doublets. The firing pattern and bursting behaviour of P10-16 CA1 neurones differed from the pattern typical of the adult. P5-9 and P10-16 cells had post-burst AHPs with a smaller amplitude and a more prolonged early phase than at late stages of development.

Sustained potassium currents in maturing CA1 hippocampal neurones

Whole-cell voltage clamp techniques were used to characterize sustained outward currents in maturing (P4 to P48) acutely isolated rat CA1 hippocampal neurones. Sodium removal and signal subtraction were used to isolate a sodium dependent sustained potassium current (IKNa). Calcium blockade (Co2+), sensitivity to a low TEA dose (0.5 mM) and sensitivity to Charibdotoxin (CTX 25 nM) and Iberiotoxin (IbTX 25 nM), in conjunction with signal subtraction, were used to isolate a sustained current with the characteristics of IC (IKCa). IKNa was found in both immature (P4-5) and older (P > 21) cells; this corresponded, respectively, to 56 +/- 5% and 36 +/- 6% of the outward current in younger and older cells. In the course of maturation, the voltage dependence of activation of IKNa shifted to more hyperpolarized values by approximately 20 mV. In the younger cells (P5-18) there was no evidence for sensitivity to CTX or IbTX. In 55 out of 77 older cells we found a component sensitive to CTX, IbTX, 0.5 mM TEA and Co2+.

Efficacy of carvedilol in reversing hypertension induced by chronic intermittent hypoxia in rats

Animal models of chronic intermittent hypoxia (CIH) mimic the hypertension observed in patients with obstructive sleep apnoea. Antihypertensive drugs were applied to these animal models to address the physiological mechanism but not to revert established hypertension. We aimed to investigate the efficacy of carvedilol (CVDL), an unselective beta-blocker that exhibits intrinsic anti-α1-adrenergic and antioxidant activities in a rat model of CIH-induced hypertension. The variability of CVDL enantiomers in plasma concentrations was also evaluated. Wistar rats with indwelling blood pressure telemeters were exposed during their sleep period to 5.6 CIH cycles/h, 10.5 h/day, for 60 days. CVDL was administered by gavage beginning on Day 36 of the CIH period and was continued for 25 days. R-(+)-CVDL and S-(-)-CVDL plasma concentrations were monitored by HPLC. CIH significantly increased diastolic and systolic blood pressure by 25.7 and 21.6 mm Hg respectively, while no effect was observed on the heart rate (HR). CVDL administration at 10, 30 and 50 mg/kg/day promoted a significant reduction in HR but did not affect arterial pressure. The S/(R+S) ratio of CVDL enantiomers was lower in rats exposed to CIH. The blockade of the sympathetic nervous system together with the putative pleiotropic effects of CVDL did not alter the CIH-induced hypertension. Although CIH induced pharmacokinetic changes in the R/(R+S) ratio, these effects do not appear to be responsible for the inability of CVDL to reverse this particular type of hypertension.

Sodium channel currents in maturing acutely isolated rat hippocampal CA1 neurones.

Sodium channel currents were recorded in excised inside-out patches from immature (P(4-10)) and older (P(20-46)) rat CA1 neurones. Channel conductance was 16.6+/-0.013 pS (P(20-46)) and 19.0+/-0.031 pS (P(4-10)). Opening patterns varied with step voltage and with age. In some patches bursting was apparent at voltages positive to -30 mV. Non-bursting behaviour was more dominant in patches from younger animals. In older animals mean open time (m.o.t.) was best described by two exponentials especially in the older cells; in the immature, there were fewer cases with two exponentials. The time constant of inactivation (tau(h)) estimated in ensemble averages was best described by two exponentials (tau(hf) and tau(hs)) in most patches from older cells. tau(hf) decreased with depolarization; tau(hs) increased in the range -30 to 0 mV. The voltage dependence of tau(hf) in the older cells is identical to that of the single tau(h) found in the younger; the results indicate a dominance of tau(hf) in the younger. Patches from younger cells more often showed one apparent active channel; in such cases, m.o.t. was described by a single exponential. However, in two cases, channels showed bursting behaviour with one of these channels showing a shift between bursting and non-bursting modes. Our findings are consistent with a heterogeneous channel population and with changes in the population in the course of maturation.

Slow and medium afterhyperpolarizations in maturing rat hippocampal CA1 neurones.

The postnatal evolution of the signals underlying the afterhyperpolarization (slow-AHP and medium-AHP) was studied in rat CA1 hippocampal neurones (P10-16, P17-23 and P greater than 26) using in vitro slices. Noradrenaline (NA) and signal subtraction were used to decompose the AHP (m-AHP and s-AHP). The amplitude of the s-AHP was found significant between P10-16 and P17-23 cells; the m-AHP showed no significant change. The time to peak of the s-AHP of the P greater than 26 cells was found to be significantly different from the remaining groups; the m-AHP showed no significant change. The changes in waveform and amplitude of the AHP at this stage were found to be mainly due to the modifications of the slow-AHP.

Dicholine succinate, the neuronal insulin sensitizer, normalizes behavior, REM sleep, hippocampal pGSK3 beta and mRNAs of NMDA receptor subunits in mouse models of depression

Central insulin receptor-mediated signaling is attracting the growing attention of researchers because of rapidly accumulating evidence implicating it in the mechanisms of plasticity, stress response, and neuropsychiatric disorders including depression. Dicholine succinate (DS), a mitochondrial complex II substrate, was shown to enhance insulin-receptor mediated signaling in neurons and is regarded as a sensitizer of the neuronal insulin receptor. Compounds enhancing neuronal insulin receptor-mediated transmission exert an antidepressant-like effect in several pre-clinical paradigms of depression; similarly, such properties for DS were found with a stress-induced anhedonia model. Here, we additionally studied the effects of DS on several variables which were ameliorated by other insulin receptor sensitizers in mice. Pre-treatment with DS of chronically stressed C57BL6 mice rescued normal contextual fear conditioning, hippocampal gene expression of NMDA receptor subunit NR2A, the NR2A/NR2B ratio and increased REM sleep rebound after acute predation. In 18-month-old C57BL6 mice, a model of elderly depression, DS restored normal sucrose preference and activated the expression of neural plasticity factors in the hippocampus as shown by Illumina microarray. Finally, young naïve DS-treated C57BL6 mice had reduced depressive- and anxiety-like behaviors and, similarly to imipramine-treated mice, preserved hippocampal levels of the phosphorylated (inactive) form of GSK3 beta that was lowered by forced swimming in pharmacologically naïve animals. Thus, DS can ameliorate behavioral and molecular outcomes under a variety of stress- and depression-related conditions. This further highlights neuronal insulin signaling as a new factor of pathogenesis and a potential pharmacotherapy of affective pathologies.

FELASA accreditation of education and training courses in laboratory animal science according to the Directive 2010/63/EU:

This document describes how the Federation of European Laboratory Animal Science Associations (FELASA) accreditation addresses both the Directive 2010/63/EU and the related European Commission guidance document. The four EU Functions and beyond: FELASA accredits courses that fulfil the requirements of Functions A, B, C and D as defined by EU Directive, Article 23, as well as for designated veterinarians and specialists in laboratory animal science. Modularity and mobility: Cohesive courses for Functions and for very specific topics are accredited, but flexibility and mobility are possible: a researcher can start his/her training with one FELASA accredited course and complete other modules with another. A course organizer will deliver a FELASA certificate relating to the successfully completed modules. Accreditation process: The process consists of two major steps: (1) a review of full course documentation provided by the applicant will lead, if successful, to FELASA accreditation. The course is posted on the FELASA website as ‘FELASA accredited’ and the course provider can deliver FELASA certificates upon successful completion of the course; (2) successful accreditation is followed by an on-site course audit. In the case of a negative outcome of the audit, FELASA accreditation is withdrawn, the course is deleted from the list of FELASA accredited courses and FELASA certificates cannot be issued. To ensure that quality is maintained, continuation of accreditation requires regular revalidation.