DEF Rats Research Articles

Profound Sex Differences in the Kidney Expression of Mitochondria-Related Genes in Endothelin-1 Receptor B (ETB) Deficient Rats Fed a High Salt Diet

Elevated levels of endothelin-1 (ET-1), overactivation of the ETA receptor and oxidative stress are critical in the development of hypertensive kidney disease. Females are more resistant than males to kidney damage; however, it is unclear if overactivation of the ETA receptor disrupts mitochondrial homeostasis similarly in both sexes. With these studies we aimed to determine if overactivation of the ET-1/ETA axis has a differential effect on mitochondrial homeostasis in the male and female kidney during high salt consumption. Male and female ETB deficient (ETB def) and transgenic (TG) control rats were placed on a high salt diet (4.0% NaCl) for 3 weeks. Kidneys were collected and the expression of 84 mitochondria-related genes in renal cortex and outer medulla was assessed with qRT-PCR arrays. In response to high salt, male ETB def rats displayed cortical overexpression of 13 genes when compared to male TG controls, while only 7 genes were upregulated in cortex of female ETB def rats compared to female TG controls. Genes upregulated in male ETB def rats are involved in apoptosis, mitochondrial biogenesis and transport, energy metabolism and ROS handling (selected genes, male ETB def vs. TG controls, n=3/group: Ucp1 (19-fold increase, p=0.03), Bnip3 and Scl25a21 (6-fold, p=0.003 and p=0.004, respectively), and Mfn1 (5-fold, p=0.03). Genes upregulated in females are involved in apoptosis, ROS handling and mitochondrial transport (selected examples, female ETB def vs. TG control, n=3/group: 2-fold increase in Sod1 (p=0.03), Akt1 (p=0.003) and Grpel1 (p=0.019). No common genes were changed between the sexes. When comparisons were made in the outer medulla, we found that deficiency of the ETB receptor in males resulted in a ≥30% reduction in the expression of 29 genes involved in apoptosis, mitochondrial biogenesis and transport, energy metabolism, ROS handling and other mitochondrial functions (p<0.05). On the other hand, the expression of 8 genes involved in mitochondrial transport and apoptosis was reduced by ≥80% in the female ETB def rat outer medulla vs. TG controls, while 3 genes involved in mitochondrial transport and energy metabolism had a 2-fold overexpression (p<0.05). Altogether, our results demonstrate that salt-sensitive hypertension affects mitochondrial homeostasis differentially in distinct regions of the kidney, and that the male and female kidney respond to salt in a disparate manner: with overactivation of the ET-1/ETA axis resulting in greater dysregulation of mitochondria-related genes in the male versus the female kidney during a high salt diet. Funded by UAB-UCSD O’Brien Center Summer Research Pathway for Students P30 DK079337 and KURE R25 DK115353 to BSM, NIGMS UAB CORD BBRT to GJC, and NIH K01HL145324 and Sapphire North America Return to Science Research grant to CDM. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Endothelin‐1 Receptor B (ETB) Deficient Rats Fed High Salt Diet Display Differential Mitochondria‐Related Gene Expression in the Kidney Cortex and Outer Medulla

Levels of the vasoactive peptide endothelin‐1 (ET‐1) are persistently elevated in patients and animal models of salt‐sensitive hypertension. Our group recently reported that loss of ETB receptor function and the consequent overactivation of the ET‐1/ETA receptor axis are associated with exaggerated kidney damage, T cell inflammation and apoptosis under high salt conditions. Importantly, the molecular mechanisms by which ET‐1 leads to high salt‐induced renal damage remain unclear. These studies aimed to determine if the overactivation of the ET‐1/ETA axis alters the kidney expression of genes involved in mitochondrial biogenesis and function during high salt consumption. Male ETB deficient (ETB def) and transgenic (TG) control rats were placed on a high salt diet (HSD, 4.0% NaCl) for 3 weeks. At the end of this HSD period, 24h urine was collected and kidneys were separated in cortex and outer medulla. Urinary protein excretion was measured as a marker of kidney damage and the kidney expression of 84 mitochondria‐related genes was assessed by qRT‐PCR arrays. Compared to TG controls, high salt‐fed ETB def rats presented with greater kidney damage as indicated by elevated protein excretion (TG control vs. ETB def, 73.9±8.0 vs. 121.4±10.0 mg/day; n=5/group; p=0.01). In addition, ETB def rats fed HSD displayed cortical overexpression of 13 genes involved in energy metabolism, apoptosis, mitochondrial biogenesis, and regulation of the production of reactive oxygen species (selected examples of ETB def vs. TG control genetic expression (n=3/group): Ucp1, 19‐fold increase (p=0.03); Bnip3 (p=0.003) and Scl25a21 (p=0.004), 6‐fold; Mfn1,5‐fold (p=0.03); and Dnm1l, 4‐fold (p=0.004)). On the other hand, when the same comparisons were made in the kidney outer medullary region, we found that deficiency of the ETB receptor resulted in a 50% reduction in the expression of 29 genes (among them, genes involved in mitochondrial membrane polarization, transport and dynamics; p<0.05). These results demonstrate a differential effect of ETAoveractivation on mitochondrial homeostasis in the kidney cortical and outer medullary regions during high salt consumption, with overactivation of the ET‐1/ETA axis leading to exaggerated mitochondrial function in the cortex and downregulation of this function in the outer medulla.

Functional Interaction of Endothelin Receptors in Mediating Natriuresis Evoked by G Protein-Coupled Estrogen Receptor 1.

The G protein–coupled estrogen receptor 1 (GPER1) mediates rapid estrogenic signaling. We recently reported that activation of GPER1 in the renal medulla evokes endothelin-1–dependent natriuresis in female, but not male, rats. However, the involvement of the ET receptors, ETA and ETB, underlying GPER1 natriuretic action remain unclear. In this study, we used genetic and pharmacologic methods to identify the contributions of ETA and ETB in mediating this female-specific natriuretic effect of renal medullary GPER1. Infusion of the GPER1-selective agonist G1 (5 pmol/kg per minute) into the renal medulla for 40 minutes increased Na+ excretion and urine flow in anesthetized female ETB-deficient (ETB def) rats and littermate controls but did not affect blood pressure or urinary K+ excretion in either group. Pretreatment with the selective ETA inhibitor ABT-627 (5 mg/kg, intravenous) abolished G1-induced natriuresis in ETB def rats. To further isolate the effects of inhibiting either receptor alone, we conducted the same experiments in anesthetized female Sprague-Dawley (SD) rats pretreated or not with ABT-627 and/or the selective ETB inhibitor A-192621 (10 mg/kg, intravenous). Neither antagonism of ETA nor antagonism of ETB receptor alone affected the G1-induced increase in Na+ excretion and urine flow in SD rats. However, simultaneous antagonism of both receptors completely abolished these effects. These data suggest that ETA and ETB receptors can mediate the natriuretic and diuretic response to renal medullary GPER1 activation in female rats.SIGNIFICANCE STATEMENTActivation of G protein–coupled estrogen receptor 1 (GPER1) in the renal medulla of female rats evokes natriuresis via endothelin receptors A and/or B, suggesting that GPER1 and endothelin signaling pathways help efficient sodium excretion in females. Thus, GPER1 activation could be potentially useful to mitigate salt sensitivity in females.

Endothelin Receptor A and B Co‐operate to Mediate GPER‐induced Natriuresis

G protein‐coupled estrogen receptors (GPER) are membrane‐associated receptors that mediate rapid estrogenic signaling. We recently found that activation of GPER in the renal medulla of female rats, but not males, evokes endothelin‐dependent natriuresis. Endothelin‐1 (ET‐1) inhibits tubular reabsorption of Na+ via stimulating ET receptor B (ETB). Evidence points that ET receptor A (ETA) may contribute to natriuresis in female rats only. The current study was designed to identify whether GPER‐induced natriuresis is mediated via activation of renal medullary ETA and/or ETBreceptors in female rats. We determined the effect of stimulating GPER in the renal medulla on urine flow and urinary Na+ excretion in anesthetized female ETB deficient rats (ETB def) and transgenic (TG) controls. We infused saline to the renal medullary interstitium during an equilibration period of 60–80 min at a flow rate of 500 μl/h, followed by vehicle, then the GPER selective agonist, G1 (5 pmol/kg/min). Infusion of G1 to the renal medulla increased urine flow and Na+excretion in TG control animals (from 4.77 ± 1.19 to 7.36 ± 2.41 μl/min and from 0.47 ± 0.09 to 0.91 ± 0.27 μmol/min, respectively, n=7, p<0.05). Similarly, medullary GPER activation evoked a diuretic and natriuretic response in ETB def rats (urine flow: from 4.07 ± 0.47 to 6.52 ± 1.01 μl/min and Na+ excretion: from 0.29 ± 0.06 to 0.64 ± 0.16 μmol/min, respectively, n=8, p<0.05). G1 infusion did not change blood pressure or urinary K+ excretion in ETB def or TG control animals. Mean arterial blood pressure was higher in ETB def rats, compared to TG controls (117 ± 6 vs. 99 ± 3 mmHg, n=7–8, p<0.05). We also determined the physiological response to medullary GPER activation in anesthetized female Sprague Dawley rats after individual blockade of ETA or ETB receptors, achieved by an intravenous bolus injection of ABT‐627 (5mg/kg) or A‐192621 (10 mg/kg), respectively, or vehicle. Neither selective antagonism of ETA nor ETB antagonism impacted the diuretic or natriuretic actions of G1. However, simultaneous blockade of ETA and ETB receptors abolished G1‐evoked diuresis and natriuresis. Blood pressure and urinary K+ excretion remained unchanged during G1 infusion in the presence or absence of ET receptor antagonists. Thus, using pharmacological and genetic approaches, our study reveals that ETA and ETB receptors in the renal medulla work co‐operatively to mediate the diuretic and natriuretic response to GPER activation.Support or Funding InformationFunded by AHA 18CDA34110010 and K99DK119413 to EYG and P01 HL136267 to DMP.

Omega-3 fatty acid deficiency impairs frontostriatal recruitment following repeated amphetamine treatment in rats: A 7 Tesla in vivo phMRI study

Although attention deficit hyperactivity disorder is associated with deficits in docosahexaenoic acid (DHA), an omega-3 fatty acid implicated in dopamine and glutamate synaptic plasticity, its role in neuroplastic brain changes that occur following repeated amphetamine (AMPH) treatment are not known. This study used pharmacological magnetic resonance imaging to investigate the impact of repeated AMPH exposure and alterations in brain DHA levels on AMPH-induced brain activation patterns. Male rats were fed a diet with no n-3 fatty acids (Deficient, DEF, n = 20), a diet fortified with preformed DHA (fish oil, FO, n = 20), or a control diet fortified with alpha-linolenic acid (n = 20) from P21 to P90. During adolescence (P40–60), one-half of each diet group received daily AMPH injections escalated weekly (0.5, 1.0, 2.5, 5.0 mg/kg/d) or drug vehicle. Following a 30-d abstinence period blood oxygen level dependent (BOLD) responses were determined in a 7 T Bruker Biospec system following an AMPH challenge (7.5 mg/kg, i.v). Postmortem erythrocyte and forebrain DHA composition were determined by gas chromatography. Compared with control rats, forebrain and erythrocyte DHA levels were significantly lower in DEF rats and significantly higher in FO rats. Across AMPH doses DEF rats exhibited greater locomotor activity compared to control and FO rats. In AMPH-naïve rats, the AMPH challenge increased BOLD activity in the substantia nigra and basal forebrain and no diet group differences were observed. In AMPH-pretreated control and FO rats, the AMPH challenge similarly increased BOLD activation in the bilateral caudate putamen, thalamus, and motor and cingulate cortices. In contrast, BOLD activation in AMPH-pretreated DEF rats was similar to AMPH-naïve DEF animals, and AMPH-pretreated DEF rats exhibited attenuated frontostriatal BOLD activation compared with AMPH-pretreated control and FO rats. These findings demonstrate that chronic escalating AMPH treatment induces enduring frontostriatal recruitment and that peri-adolescent deficits in brain DHA accrual impair this response.

Deficits in Docosahexaenoic Acid Accrual during Adolescence Reduce Rat Forebrain White Matter Microstructural Integrity: An in vivo Diffusion Tensor Imaging Study

Neuropsychiatric disorders that frequently initially emerge during adolescence are associated with deficits in the omega-3 (n-3) fatty acid docosahexaenoic acid (DHA), elevated proinflammatory signaling, and regional reductions in white matter integrity (WMI). This study determined the effects of altering brain DHA accrual during adolescence on WMI in the rat brain by diffusion tensor imaging (DTI), and investigated the potential mediating role of proinflammatory signaling. During periadolescent development, male rats were fed a diet deficient in n-3 fatty acids (DEF, n = 20), a fish oil-fortified diet containing preformed DHA (FO, n = 20), or a control diet (CON, n = 20). In adulthood, DTI scans were performed and brain WMI was determined using voxelwise tract-based spatial statistics (TBSS). Postmortem fatty acid composition, peripheral (plasma IL-1β, IL-6, and C-reactive protein [CRP]) and central (IL-1β and CD11b mRNA) proinflammatory markers, and myelin basic protein (MBP) mRNA expression were determined. Compared with CON rats, forebrain DHA levels were lower in DEF rats and higher in FO rats. Compared with CON rats, DEF rats exhibited greater radial diffusivity (RD) and mean diffusivity in the right external capsule, and greater axial diffusivity in the corpus callosum genu and left external capsule. DEF rats also exhibited greater RD than FO rats in the right external capsule. Forebrain MBP expression did not differ between groups. Compared with CON rats, central (IL-1β and CD11b) and peripheral (IL-1β and IL-6) proinflammatory markers were not different in DEF rats, and DEF rats exhibited lower CRP levels. These findings demonstrate that deficits in adolescent DHA accrual negatively impact forebrain WMI, independently of elevated proinflammatory signaling.

Glutamate homeostasis in the adult rat prefrontal cortex is altered by cortical docosahexaenoic acid accrual during adolescence: An in vivo1H MRS study

Major psychiatric disorders are associated with dysregulated glutamate homeostasis and deficits in the omega-3 fatty acid docosahexaenoic acid (DHA). This study determined the effects of dietary-induced alterations in brain DHA accrual on cortical glutamate homeostasis in the adult rat brain. Adolescent rats were fed a control diet (n = 20), a n-3 fatty acid-deficient diet (DEF, n = 20), or a fish oil-fortified diet containing preformed DHA (FO, n = 20). In adulthood 1H MRS scans were performed with voxels in the prefrontal cortex (PFC) and thalamus. Compared with controls, erythrocyte, PFC, and thalamus DHA levels were significantly lower in DEF rats and significantly higher in FO rats. In the PFC, but not the thalamus, glutamate was significantly elevated in DEF rats compared with controls and FO rats. Glutamine did not differ between groups and the glutamine/glutamate ratio was lower in DEF rats. No differences were observed for markers of excitotoxicity (NAA, GFAP), or astrocyte glutamate transporter (GLAST, GLT-1) or glutamine synthetase expression. Across diet groups, PFC DHA levels were inversely correlated with PFC glutamate levels and positively correlated with GLAST expression. Together these findings demonstrate that rat cortical DHA accrual during adolescence impacts glutamate homeostasis in the adult PFC.

Endothelin receptor-specific control of endoplasmic reticulum stress and apoptosis in the kidney

Endothelin-1 (ET-1) promotes renal damage during cardiovascular disease; yet, the molecular mechanisms involved remain unknown. Endoplasmic reticulum (ER) stress, triggered by unfolded protein accumulation in the ER, contributes to apoptosis and organ injury. These studies aimed to determine whether the ET-1 system promotes renal ER stress development in response to tunicamycin. ETB deficient (ETB def) or transgenic control (TG-con) rats were used in the presence or absence of ETA receptor antagonism. Tunicamycin treatment similarly increased cortical ER stress markers in both rat genotypes; however, only ETB def rats showed a 14–24 fold increase from baseline for medullary GRP78, sXBP-1, and CHOP. Pre-treatment of TG-con rats with the ETA blocker ABT-627 for 1 week prior to tunicamycin injection significantly reduced the ER stress response in cortex and medulla, and also inhibited renal apoptosis. Pre-treatment with ABT-627 failed to decrease renal ER stress and apoptosis in ETB def rats. In conclusion, the ET-1 system is important for the development of tunicamycin-induced renal ER stress and apoptosis. ETA receptor activation induces renal ER stress genes and apoptosis, while functional activation of the ETB receptor has protective effects. These results highlight targeting the ETA receptor as a therapeutic approach against ER stress-induced kidney injury.

Loss of endothelin B receptor function impairs sodium excretion in a time- and sex-dependent manner.

Recent studies suggested a direct link between circadian rhythms and regulation of sodium excretion. Endothelin-1 (ET-1) regulates sodium balance by promoting natriuresis through the endothelin B receptor (ETB) in response to increased salt in the diet, but the effect that the time of day has on this natriuretic response is not known. Therefore, this study was designed to test the hypothesis that ETB receptor activation contributes to the diurnal control of sodium excretion and that sex differences contribute to this control as well. Twelve-hour urine collections were used to measure sodium excretion. On day 3 of the experiment, a NaCl load (900 μeq) was given by oral gavage either at Zeitgeber time [ZT] 0 (inactive period) or ZT12 (active period) to examine the natriuretic response to the acute salt load. Male and female ETB-deficient (ETB def) rats showed an impaired natriuretic response to a salt load at ZT0 compared with their respective transgenic controls (Tg cont). Male ETB def rats showed a delayed natriuretic response to a salt load given at ZT12 compared with male Tg cont, a contrast to the prompt response shown by female ETB def rats. Treatment with ABT-627, an ETA receptor antagonist, improved the natriuretic response seen within the first 12 h of a ZT0 salt load in both sexes. These findings demonstrate that diurnal excretion of an acute salt load 1) requires ET-1 and the ETB receptor, 2) is more evident in male vs. female rats, and 3) is opposed by the ETA receptor.

Abstract P639: Attenuation of Renal Inner Medullary Circadian Clock Gene Expression in Response to High Salt Intake is Dependent on the Endothelin B Receptor

Our lab has recently shown that ETB deficient (ETB def) rats have a time of day dependent impairment in their ability to excrete a Na+ load. These observations suggest an interaction between renal ETB receptors and circadian mechanisms that regulate renal tubular Na+ transport and excretion. Given that knockout of the circadian clock gene Bmal1 reduces blood pressure in mice, we hypothesized that a high salt intake impairs the clock mechanism in the renal inner medulla in an ETB dependent manner. Transgenic control (Tg con) or ETB def rats were fed normal (NS, 0.8% NaCl) or high (HS, 4% NaCl) salt for two weeks. In one group, rats were euthanized every 4 hours beginning at zeitgeber time 0 (lights on) for tissue collection (and subsequent assessment of circadian clock genes), while in a second group of rats urine was collected in 12-hour intervals (active vs. inactive). Consistent with our hypothesis, we observed that HS abolished the normal oscillation in Bmal1 expression in the renal inner medulla of Tg con rats, and effect not observed in ETB def rats. Interestingly, renal production of ET-1, was significantly higher during the active period vs. inactive period in both NS (3.6±1.1 vs. 0.8±0.2 pg/12hr respectively) and HS (9.2±4.1 vs. 1.6±0.3 pg/12hr respectively) fed Tg con rats. There was no time-of-day-dependent difference in ET-1 excretion in ETB def rats on NS (6.6±2.2 vs. 4.6±1.7 pg/12hr respectively), although this pattern was restored in ETB def rats fed HS (2.2±1.0 vs. 9.2±2.5 pg/12hr inactive vs. active). Taken together, these data indicate that an increase in renal ET-1/ETB activation in response to HS modulates inner medullary clock gene expression to promote renal Na+ excretion.

Sex Differences in the Diurnal Natriuretic Response to a Salt Load in Rats Lacking a Functional ETB Receptor

Male rats lacking the ETB receptor (ETB def) exhibit salt‐sensitive hypertension due to impaired renal sodium handling. Given that females generally have a reduced incidence of salt‐sensitive hypertension compared to males, we hypothesized that female ETB def rats are better able to excrete a salt load compared to males. ETB def and genetic control rats of both sexes were implanted with telemetry transmitters to monitor mean arterial pressure (MAP) and given at least a week to recover. Following several days of baseline urine collection in 12 hr light/dark increments, rats were given a single 900µEq Na salt load (NaCl) by oral gavage at the beginning of their active (7pm‐7am, dark) or inactive (7am‐7pm, light) period. Urine was again collected in 12 hr intervals for several more days. We previously showed that ETB def males excrete significantly less salt during the initial 12 hr following an acute salt load whether given at the beginning of the active or inactive period compared to genetic controls, an effect that was more pronounced when the salt load was given prior to the inactive period. ETB def females excreted significantly more salt vs. ETB def males following the acute salt load given during the beginning of the active period (673 ± 111 vs. 320 ± 98µEq Na/12hr, P<0.05) and again showed an improved natriuresis vs. ETB def males following a salt load given at the start of the inactive period (183 ± 60 vs. 50 ± 91µEq Na/12hr). There was no significant effect of the salt loading on MAP in any group, although both male and female ETB def rats had significantly elevated MAP compared to controls. These data show that despite the loss of the ETB receptor, females excrete an acute salt load more efficiently than males.

High salt diet increases the pressor response to stress in female, but not male ETB-receptor-deficient rats.

Acute stress in both rodents and humans causes a transient rise in blood pressure associated with an increase in plasma endothelin-1 (ET-1). High salt (HS) intake also increases ET-1 production, and interestingly, blunts the pressor response to acute air jet stress in rats. We previously reported that female rats lacking functional ETB receptors everywhere except sympathetic nerves (ETB def) had a greater degree of hypertension in response to a HS diet compared to their male counterparts when measured by the tail cuff method. However, we now report that salt-induced hypertension is not different between sexes when measured by telemetry. Therefore, additional experiments were designed to test the hypothesis that female ETB def rats are more sensitive to acute stress when on a HS diet. The pressor response, measured by telemetry, to acute air jet stress was similar between male transgenic control (Tg control) and ETB def rats following chronic HS intake. In contrast, female ETB def rats had a significantly greater pressor response (about twofold higher) than female or male Tg control or male ETB def rats maintained on HS, a finding that cannot be explained by increased vascular reactivity to ET-1 in female rats as we observed that male ETB def rats had a greater pressor response to i.v. infusion of ET-1 compared to females. Furthermore, HS feeding exacerbated the pressor response to ET-1 in both male and female ETB def rats. Given our previous studies demonstrating that the ETA receptor functions to reduce the pressor response to acute stress, these findings further support a role for the ET receptor system in the pressor response to acute stress and that female rats have reduced ETA receptor activity when on a HS diet compared to males.

Omega-3 fatty acid deficient male rats exhibit abnormal behavioral activation in the forced swim test following chronic fluoxetine treatment: Association with altered 5-HT1A and alpha2A adrenergic receptor expression

Omega-3 fatty acid deficiency during development leads to enduing alterations in central monoamine neurotransmission in rat brain. Here we investigated the effects of omega-3 fatty acid deficiency on behavioral and neurochemical responses to chronic fluoxetine (FLX) treatment. Male rats were fed diets with (CON, n = 34) or without (DEF, n = 30) the omega-3 fatty acid precursor alpha-linolenic acid (ALA) during peri-adolescent development (P21–P90). A subset of CON (n = 14) and DEF (n = 12) rats were administered FLX (10 mg/kg/d) through their drinking water for 30 d beginning on P60. The forced swimming test (FST) was initiated on P90, and regional brain mRNA markers of serotonin and noradrenaline neurotransmission were determined. Dietary ALA depletion led to significant reductions in frontal cortex docosahexaenoic acid (DHA, 22:6n-3) composition in DEF (−26%, p = 0.0001) and DEF + FLX (−32%, p = 0.0001) rats. Plasma FLX and norfluoxetine concentrations did not different between FLX-treated DEF and CON rats. During the 15-min FST pretest, DEF + FLX rats exhibited significantly greater climbing behavior compared with CON + FLX rats. During the 5-min test trial, FLX treatment reduced immobility and increased swimming in CON and DEF rats, and only DEF + FLX rats exhibited significant elevations in climbing behavior. DEF + FLX rats exhibited greater midbrain, and lower frontal cortex, 5-HT1A mRNA expression compared with all groups including CON + FLX rats. DEF + FLX rats also exhibited greater midbrain alpha2A adrenergic receptor mRNA expression which was positively correlated with climbing behavior in the FST. These preclinical data demonstrate that low omega-3 fatty acid status leads to abnormal behavioral and neurochemical responses to chronic FLX treatment in male rats.

Omega-3 fatty acid deficiency does not alter the effects of chronic fluoxetine treatment on central serotonin turnover or behavior in the forced swim test in female rats

While translational evidence suggests that long-chain omega-3 fatty acid status is positively associated with the efficacy of selective serotonin reuptake inhibitor drugs, the neurochemical mechanisms mediating this interaction are not known. Here, we investigated the effects of dietary omega-3 (n-3) fatty acid insufficiency on the neurochemical and behavioral effects of chronic fluoxetine (FLX) treatment. Female rats were fed diets with (CON, n=56) or without (DEF, n=40) the n-3 fatty acids during peri-adolescent development (P21–P90), and one half of each group was administered FLX (10mg/kg/day) for 30days (P60–P90) prior to testing. In adulthood (P90), regional brain serotonin (5-HT) and 5-hydroxyindoleacetic (5-HIAA) concentrations, presynaptic markers of 5-HT neurotransmission, behavioral responses in the forced swim test (FST), and plasma FLX and norfluoxetine (NFLX) concentrations were investigated. Peri-adolescent n-3 insufficiency led to significant reductions in cortical docosahexaenoic acid (DHA, 22:6n-3) composition in DEF (−25%, p≤0.0001) and DEF+FLX (−28%, p≤0.0001) rats. Untreated DEF rats exhibited significantly lower regional 5-HIAA/5-HT ratios compared with untreated CON rats, but exhibited similar behavioral responses in the FST. In both CON and DEF rats, chronic FLX treatment similarly and significantly decreased 5-HIAA concentrations and the 5-HIAA/5-HT ratio in the hypothalamus, hippocampus, and nucleus accumbens, brainstem tryptophan hydroxylase-2 mRNA expression, and immobility in the FST. While the FLX-induced reduction in 5-HIAA concentrations in the prefrontal cortex was significantly blunted in DEF rats, the reduction in the 5-HIAA/5-HT ratio was similar to CON rats. Although plasma FLX and NFLX levels were not significantly different in DEF and CON rats, the NFLX/FLX ratio was significantly lower in DEF+FLX rats. These preclinical data demonstrate that n-3 fatty acid deficiency does not significantly reduce the effects of chronic FLX treatment on central 5-HT turnover or behavior in the FST in female rats.

Prenatal choline deficiency does not enhance hippocampal vulnerability after kainic acid-induced seizures in adulthood

Choline is a vital nutrient needed during early development for both humans and rodents. Severe dietary choline deficiency during pregnancy leads to birth defects, while more limited deficiency during mid- to late pregnancy causes deficits in hippocampal plasticity in adult rodent offspring that are accompanied by cognitive deficits only when task demands are high. Because prenatal choline supplementation confers neuroprotection of the adult hippocampus against a variety of neural insults and aids memory, we hypothesized that prenatal choline deficiency may enhance vulnerability to neural injury. To examine this, adult offspring of rat dams either fed a control diet (CON) or one deficient in choline (DEF) during embryonic days 12–17 were given multiple injections (i.p.) of saline (control) or kainic acid to induce seizures and were euthanized 16days later. Perhaps somewhat surprisingly, DEF rats were not more susceptible to seizure induction and showed similar levels of seizure-induced hippocampal histopathology, GAD expression loss, upregulated hippocampal GFAP and growth factor expression, and increased dentate cell and neuronal proliferation as that seen in CON rats. Although prenatal choline deficiency compromises adult hippocampal plasticity in the intact brain, it does not appear to exacerbate the neuropathological response to seizures in the adult hippocampus at least shortly after excitotoxic injury.

Prenatal choline availability modulates hippocampal neurogenesis and neurogenic responses to enriching experiences in adult female rats

Increased dietary intake of choline early in life improves performance of adult rats on memory tasks and prevents their age-related memory decline. Because neurogenesis in the adult hippocampus also declines with age, we investigated whether prenatal choline availability affects hippocampal neurogenesis in adult Sprague-Dawley rats and modifies their neurogenic response to environmental stimulation. On embryonic days (ED) 12-17, pregnant rats ate a choline-supplemented (SUP-5 g/kg), choline sufficient (SFF-1.1 g/kg), or choline-free (DEF) semisynthetic diet. Adult offspring either remained in standard housing or were given 21 daily visits to explore a maze. On the last ten exploration days, all rats received daily injections of 5-bromo-2-deoxyuridine (BrdU, 100 mg/kg). The number of BrdU+ cells was significantly greater in the dentate gyrus in SUP rats compared to SFF or DEF rats. While maze experience increased the number of BrdU+ cells in SFF rats to the level seen in the SUP rats, this enriching experience did not alter cell proliferation in DEF rats. Similar patterns of cell proliferation were obtained with immunohistochemical staining for neuronal marker doublecortin, confirming that diet and exploration affected hippocampal neurogenesis. Moreover, hippocampal levels of the brain-derived neurotrophic factor (BDNF) were increased in SUP rats as compared to SFF and DEF animals. We conclude that prenatal choline intake has enduring effects on adult hippocampal neurogenesis, possibly via up-regulation of BDNF levels, and suggest that these alterations of neurogenesis may contribute to the mechanism of life-long changes in cognitive function governed by the availability of choline during gestation.

N‐3 Fatty Acid Deficient Diet Affects Rat Spatial Performance in the Barnes Circular Maze.

Our previous work demonstrated that a decrease in brain docosahexaenoic acid (DHA) in rodents was associated with poorer performance in the Morris water maze. In this study we showed a deficit in spatial task performance of n-3 deficient rats using the Barnes circular maze, which is similar to the Morris maze in that both tests require an escape response. Deficiency has been accomplished through the use of a two generational model in which the dam is deprived of n-3 fatty acid sources and her offspring are then weaned to the same deficient diet. There was a loss of 56% of total brain DHA in n-3 deficient (n-3 Def) in comparison to n-3 adequate (n-3 Adq) rats. Both n-3 Def (n=11) and n-3 Adq (n=10) rats learned to locate the escape tunnel during the four days of training (2 trials a day), as indicated by a progressive reduction in escape latencies and errors rate. However, during the first two days, n-3 Def rats did not perform as well as n-3 Adq, but differences between groups did not reach the significance. To evaluate memory retention, rats were retested in the Barnes maze ten days after the last training trial. In the retention testing, n-3 Adq rats did not differ from n-3 Def in escape latencies (27.3+6.0 sec vs 24.9+7.2 sec)) and errors rate (4.55+1.2 vs 3.80+1.1). One week after memory retention testing, the escape tunnel was moved to a new position (opposite to the original), and rats were retrained in five consecutive trials to find the new location of the target (reversal learning). Def rats required more time to find the new position of the escape tunnel (p=0.003). This increase in the escape latencies was accompanied by a significantly higher number of errors. So, this study demonstrated a deficit in spatial task performance rats with low brain DHA in the two generational model. This project was funded by the Intramural Research Program of the NIAAA/NIH.

Subtle abnormalities of gait detected early in vitamin B6 deficiency in aged and weanling rats with hind leg gait analysis.

Motor abnormalities have been observed in every species made vitamin B6 deficient, and have been detected and quantified early in vitamin B6 deficiency in young adult female Long-Evans rats with hind leg gait analysis. Our objective was to determine if hind leg gait analysis could be used to detect vitamin B6 deficiency in weanling (3 weeks) and aged (23 months) Fischer 344 male rats. Rats (n = 10 per group) were fed: the control diet ad libitum (AL-CON); the control diet devoid of added pyridoxine hydrochloride (DEF); or the control diet pair-fed to DEF (PF-CON). At 10 weeks, plasma pyridoxal phosphate concentration confirmed deficiency in both age groups. Gait abnormalities were detected in the absence of gross motor disturbances in both aged and weanling DEF rats at 2-3 weeks. Width of step was significantly reduced (16%, p less than 0.003) in DEF aged rats compared to AL- and PF-CON. This pattern of response was similar to that reported previously in young adult rats. In weanling rats, pair feeding alone reduced mean width of step (+/- SEM) by 25% compared to ad libitum feeding (2.7 +/- 0.1 vs 3.6 +/- 0.1 cm for PF- vs AL-CON, respectively, p less than 0.05). In DEF weanling rats, width (3.0 +/- 0.1 cm) was increased compared to PF-CON (11%, p less than 0.05) but decreased compared to AL-CON (16%, p less than 0.05). Width of step was significantly altered early in B6 deficiency in rats of different ages and strains and in both sexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Supplemental effect of retinoids on immume function in vitamin a deficient rats

To investigate the effect of retinoids on immune function, retinyl acetate(A), retinoic acid(RA) and methyl 5,6-epoxyretinoate(Epo-A) were injected to vitamin A deficient (A DEF) rats and their effects were compared with that of A DEF rats. PHA skin test was carried out during the period of retinoid injection(1 μg/day). After the retinoid injection of 100–500 μg i.m., blood was drawn for measuring serum protein fractions, and the spleen was investigated. Reactivity to PHA was increased after the injection of A, RA and Epo-A. Serum IgG levels were elevated in Epo-A group. Spleen weight and its mononuclear cell count increased after the injection of A, RA, and Epo-A. Histologically, the periarterial sheath was attenuated, packed with small lymphocytes, and the germinal center was rudimentary in A DEF rats. In the A and RA injected group, on the other hand, lymphatic nodules with well developed germinal centers were present and large, clear cells were found diffusely around the periarterial region. These findings suggest that A and RA may act to maintain well developed B cells in A DEF rats. Furthermore, it is suggested that Epo-A can also act to enhance the immune function of A DEF rats.

The Effect of Vitamin A Deficiency on the in Vitro Cellular Immune Response of Rats

The effect of vitamin A deficiency on the response of splenic lymphocytes to mitogenic stimulation was determined in an experimental rat model. Male Lewis rats were divided into three groups. The ad libitum group (AL) was fed unlimited amounts of a vitamin A-supplemented diet. The vitamin A-deficient group (DEF) received a commercial vitamin A-free diet. The pair-fed group (PF) received a vitamin A-containing diet equivalent in amount to that consumed by the DEP group. During the early stages of vitamin A deficiency (determined by cessation of weight gain), the rats were killed and the isolated splenic lymphocytes subjected to mitogenic stimulation. Lymphocytes from DEF rats had one-third the transformation response to the mitogens Concanavalin A, Phytohemagglutinin and E. coli Lipopolysaccharide S of the AL and PF groups. When the DEF rats were supplemented with vitamin A, the transformation response returned to control values within 3 days. In addition to the alterations in the immune response, the DEF rats showed a marked leukopenia, a decrease in the number of circulating lymphocytes and an increase in the number of circulating neutrophils.