TP Rats Research Articles

The Rewarding and Anxiolytic Properties of Ethanol within the Central Nucleus of the Amygdala: Mediated by Genetic Background and Nociceptin.

In humans, alcohol is consumed for its rewarding and anxiolytic effects. The central nucleus of the amygdala (CeA) is considered a neuronal nexus that regulates fear, anxiety, and drug self-administration. Manipulations of the CeA alter ethanol (EtOH) consumption under numerous EtOH self-administration models. The experiments determined whether EtOH is reinforcing/anxiolytic within the CeA, whether selective breeding for high alcohol consumption alters the rewarding properties of EtOH in the CeA, and whether the reinforcing/anxiolytic effects of EtOH in the CeA are mediated by the neuropeptides corticotropin-releasing factor (CRF) and nociceptin. The reinforcing properties of EtOH were determined by having male Wistar and Taconic alcohol-preferring (tP) rats self-administer EtOH directly into the CeA. The expression of anxiety-like behaviors was assessed through multiple behavioral models (social interaction, acoustic startle, and open field). Coadministration of EtOH and a CRF1 antagonist (NBI35965) or nociceptin on self-administration into the CeA and anxiety-like behaviors was determined. EtOH was self-administered directly into the lateral CeA, and tP rats self-administered a lower concentration of EtOH than Wistar rats. EtOH microinjected into the lateral CeA reduced the expression of anxiety-like behaviors, indicating an anxiolytic effect. Coadministration of NBI35965 failed to alter the rewarding/anxiolytic properties of EtOH in the CeA. In contrast, coadministration of the nociceptin enhanced both EtOH reward and anxiolysis in the CeA. Overall, the data indicate that the lateral CeA is a key anatomic location that mediates the rewarding and anxiolytic effects of EtOH, and local nociceptin receptors, but not local CRF1 receptors, are involved in these behaviors. SIGNIFICANCE STATEMENT: Alcohol is consumed for the stimulatory, rewarding, and anxiolytic properties of the drug of abuse. The current data are the first to establish that alcohol is reinforcing and anxiolytic within the lateral central nucleus of the amygdala (CeA) and that the nociceptin system regulates these effects of alcohol within the CeA.

Placental Ischemia‐Stimulated TH17 Cells Induce Preeclampsia‐Associated Cytolytic Natural Killer Cells During Pregnancy

The Reduced Uterine Perfusion Pressure (RUPP) rat model of placental ischemia mimics many of the characteristics of preeclampsia (PE) including hypertension, intrauterine growth restriction, and increases in circulating TH17 cells (TH17s), IL‐17 and placental cytolytic natural killer cells (cNKs). Although the stimulus for cNKs in PE is currently unknown, recent in vitro studies suggest that IL‐17 induces proliferation and cytolytic activity in human NK cells. We have previously demonstrated that placental ischemia (PI)‐stimulated TH17s mediate pathophysiology in pregnant rats similar to that seen in PE. In this study we tested the hypothesis that PI‐stimulated TH17s induce cNKs and their associated cytokines (IFNγ, TNFα) and cytolytic proteins (perforin, granzymes). We further hypothesized that inhibition of oxidative stress (ROS) with Tempol (Tp) would attenuate NK cytolytic activation. PI‐stimulated TH17s were isolated from RUPP and injected i.p. into normal pregnant (NP) rats on gestation day (GD) 12 with or without Tp administration ad libitum in drinking water (GD 12–19). Circulating TH17s and total NK cells (tNKs) and cNKs in placentas were quantified via flow cytometry in NP, NP+TH17, and NP+TH17+Tp rats. MAP, pup weight, placental ROS, cytokines, perforin and granzymes were measured. Circulating TH17s and IL‐17 were significantly increased after TH17 adoptive transfer. Circulating TH17s (% gated): NP (n=7) − 1.2±0.3%, NP+TH17 (n=7) − 4.2±0.8%, NP+TH17+Tp (n=7) − 0.8±0.2% (p<0.05 vs NP+TH17). MAP increased from 96.3±4.7 mmHg in NP to 117.9±2.2 mmHg in NP+TH17, and was normalized to 102±2.6 mmHg in NP+TH17+Tp (p<0.05 vs NP+TH17s). Placental tNKs (% gated) were significantly increased in NP+TH17 (19.7±4.6%) compared to NP (5.4±2.0%) and NP+TH17+Tp (3.1±1.3%; p<0.05 vs NP+TH17). Importantly, placental cNKs were significantly increased in response to TH17s (NP: 2.9±0.9% vs NP+TH17: 14.9 ±4.0%) and were inhibited with Tp treatment (2.8±1.0%; p<0.05 vs NP+TH17). Pup weight decreased from 2.4±0.04 mg in NP to 1.9±0.1 mg in NP+TH17 and increased to 2.3±0.04 mg in NP+TH17+Tp (p<0.05 vs NP+TH17). Placental ROS increased from 223.5±63 RLU in NP to 574.3±95.3 RLU in NP+TH17 and decreased to 247.8±71.5 RLU in NP+TH17+Tp (p<0.05 vs NP+TH17). Placental inflammation was significantly increased in response to PI‐stimulated TH17s and was attenuated with Tp treatment. Placental IFNγ: NP − 127.4±4.0 pg/mg, NP+TH17 − 153±16.6 pg/mg, NP+TH17+Tp − 116±4.0 pg/mg (p<0.05 vs NP+TH17); Placental TNFα: NP − 76.3±9.2 pg/mg, NP+TH17 − 129.8±11.1 pg/mg, NP+TH17+Tp − 61.5±21.1 pg/mg (p< 0.05 vs NP+TH17). Placental NK cytolytic proteins were significantly increased in response to PI‐stimulated TH17s. Placental perforin: NP − 0.3±0.2 pg/mg NP+TH17 − 1.6±0.2 pg/mg, NP+TH17+Tp − 1.2±0.7 pg/mg in (p<0.05 vs NP); Placental Granzyme A: NP − 91.6±18 pg/mg, NP+TH17 − 180.6±26.6 pg/mg, NP+TH17+Tp − 399.7±54.7 mg/pg (p<0.05 vs NP). These data suggest a role for PI‐stimulated TH17s to induce a cytotoxic phenotype in NKs during pregnancy and identifies stimulation of cNKs as a new mechanism by which TH17s may mediate PE pathophysiology.Support or Funding InformationNIH: HL130456, HD067541This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neonatal androgenization-induced early endocrine-metabolic and ovary misprogramming in the female rat.

Androgen excess predisposes the organism to develop metabolic-endocrine and reproductive dysfunctions, among them the development of a phenotype resembling that of human Polycystic Ovary Syndrome (PCOS). We analyzed the impact of a single neonatal (5day-old) testosterone propionate (TP; s.c. 1.25mg/female pup) dose on: a) several metabolic-endocrine activities and b) ovarian steroidogenic and granulosa cell (GC) functions and also follicular population in juvenile and adult TP and control (CT) rats. Compared to CT rats, TP animals were characterized by: a) accelerated growth, hyperadiposity and hyperleptinemia, b) very early (pre-weaning age) vaginal opening, c) hyperinsulinemia in adult life, d) dysfunctional ovarian steroidogenesis, e) conserved GC functionality in both juveniles (in vitro) and adults (in vivo), and f) estrous cycles arrested at estrus. Finally, histological studies of the ovaries indicated that in TP (vs. CT) rats: i) primary and antral follicle frequencies were 3- and 15-fold higher and lower, respectively, in juveniles and ii) secondary and atretic follicle frequencies were 3- and 5-fold lower and higher, respectively, in adults. Large cystic images without corpus luteum were observed in the ovaries from adult TP rats only. Our results strongly suggest that transient neonatal hyperandrogenemia induced early misprogramming of metabolic-endocrine and ovarian (steroidogenesis/folliculogenesis) functions. Conversely, TP rats preserved their ovary GC endocrine function. Our results further support the high risk of developing ovarian hyperstimulation syndrome for infertile women with transient/chronic hyperandrogenemia (PCOS) subjected to assisted reproductive technologies.

Parametrial Adipose Tissue and Metabolic Dysfunctions Induced by Fructose‐rich Diet in Normal and Neonatal‐androgenized Adult Female Rats

Hyperandrogenemia predisposes an organism toward developing impaired insulin sensitivity. The aim of our study was to evaluate endocrine and metabolic effects during early allostasis induced by a fructose-rich diet (FRD) in normal (control; CT) and neonatal-androgenized (testosterone propionate; TP) female adult rats. CT and TP rats were fed either a normal diet (ND) or an FRD for 3 weeks immediately before the day of study, which was at age 100 days. Energy intake, body weight (BW), parametrial (PM) fat characteristics, and endocrine/metabolic biomarkers were then evaluated. Daily energy intake was similar in CT and TP rats regardless of the differences in diet. When compared with CT-ND rats, the TP-ND rats were heavier, had larger PM fat, and were characterized by basal hypoadiponectinemia and enhanced plasma levels of non-esterified fatty acid (NEFA), plasminogen activator inhibitor-1 (PAI-1), and leptin. FRD-fed CT rats, when compared with CT-ND rats, had high plasma levels of NEFA, triglyceride (TG), PAI-1, leptin, and adiponectin. The TP-FRD rats, when compared with TP-ND rats, displayed enhanced leptinemia and triglyceridemia, and were hyperinsulinemic, with glucose intolerance. The PM fat taken from TP rats displayed increase in the size of adipocytes, decrease in adiponectin (protein/gene), and a greater abundance of the leptin gene. PM adipocyte response to insulin was impaired in CT-FRD, TP-ND, and TP-FRD rats. A very short duration of isocaloric FRD intake in TP rats induced severe metabolic dysfunction at the reproductive age. Our study supports the hypothesis that the early-androgenized female rat phenotype is highly susceptible to developing endocrine/metabolic dysfunction. In turn, these abnormalities enhance the risk of metabolic syndrome, obesity, type 2 diabetes, and cardiovascular disease.

Pumpkin Seed Oil and Phytosterol-F Can Block Testosterone/Prazosin-Induced Prostate Growth in Rats

Introduction: This study was undertaken to investigate the effects of pumpkin seed oil alone or combined with Phytosterol-F on testosterone/prazosin-induced (T-P) prostate growth in rats. Materials and Methods: Forty adult Wistar rats were divided into five groups, including: one control group, rats treated with vehicle only, one group treated with T-P, and two groups of T-P-treated rats, one receiving orally pumpkin seed oil alone and one group receiving orally pumpkin seed oil combined with Phytosterol-F. Two weeks later, the prostatic weight-to-body weight ratio was determined after sacrifice. The total protein concentration was measured by using a protein assay. Some ventral prostatic tissues were histologically examined after hematoxylin-eosin staining. Results: Histological sections of the ventral prostate showed that the architecture of the prostate glands became hyperplastic in the T-P rats, but not in the control or vehicle-treated animals. As compared with the control or vehicle group, T-P rats had a significantly higher prostatic weight-to-body weight ratio for the ventral prostate (p = 0.05 and p = 0.007, respectively), but not for the dorsolateral prostate (p = 0.53 and p = 0.73, respectively). The T-P rats had significantly higher protein levels within both lobes (ventral lobe, p = 0.02 and p < 0.0001, respectively; dorsolateral lobe, p = 0.06 and p = 0.005, respectively). As compared with the T-P-alone rats, the TP rats treated with pumpkin seed oil alone or pumpkin seed oil combined with Phytosterol-F had a significantly lower weight ratio for the ventral prostate (p = 0.01 and p = 0.004, respectively) and significantly lower protein levels within both lobes (p = 0.03 and p = 0.003, respectively; p = 0.007 and p = 0.002, respectively). In addition, Phytosterol-F had some additive effect on the total protein synthesis within the ventral prostate (p = 0.02). Conclusion: Pumpkin seed oil alone or combined with Phytosterol-F can block the T-P-induced increases in prostatic weight-to-body weight ratio and protein synthesis.

EVIDENCE OF APOPTOSIS IN THE CASTRATION-INDUCED ATROPHY OF THE RAT LEVATOR ANI MUSCLE

Apoptosis or programmed cell death has been previously reported in androgen-responsive tissue such as the prostate. We tested the hypothesis that apoptosis may also represent a component of the castration-induced atrophy of the sexually dimorphic levator ani (LA) muscle of the male rat. Gonadectomy (GDX) induced a severe decrease in the LA muscle wet weight accompanied by important modifications in cytoarchitecture including an increase in myofibrillar interspace and condensed mitochondria. These alterations were almost completely reversed after 7 days of testosterone propionate replacement therapy (GDX + TP). In GDX rats, internucleosomal DNA fragmentation was confirmed by both agarose gel electrophoresis and electron microscopy. DNA fragmentation was no longer detected in GDX + TP rats. In GDX rats, overexpression of the trpm-2/clusterin gene, used as an early marker of apoptosis, further confirmed that an apoptotic response, typical of androgen-responsive tissues, is taking place following androgen withdrawal.

Androgen receptors in cranial nerve motor nuclei of male and female rats

This study compared AR proteins in four cranial nerve motor nuclei among male and female rats that were intact, gonadectomized, or gonadectomized and given TP by immunohistochemistry. AR-immunoreactive (ir) neurons were found, in descending order of abundance, in the nucleus ambiguus, hypoglossal nucleus, and the facial and trigeminal motor nuclei of both males and females of intact and gonadectomized plus TP rats. Virtually every neuron of the nucleus ambiguus was AR-ir. In contrast, AR-ir neurons were either restricted to a specific area of the hypoglossal nucleus, or randomly distributed in the facial and trigeminal motor nuclei. The predominant AR-ir site shifted from cell nuclei to the cytoplasm, depending upon the presence or absence of ligand. Sex differences in the amount and staining intensity of AR-ir neurons were discernable in all four motor nuclei of intact rats, and these differences were maintained in gonadectomized plus TP rats, with the exception of the nucleus ambiguus. The immunostaining results were complemented by results from AR binding studies. Cytosolic AR binding values for the hypoglossal and facial motor nuclei of females were only approximately 50% of those of males despite the absence of a sex difference in neuron number. These results indicate that intrinsic sex differences in AR levels and androgenic regulation of AR exist in cranial nerve motor nuclei, and that there are differences in the abundance and distribution pattern of AR responsive neurons in cranial nerve motor nuclei. These results are consistent with the idea that sex differences in AR could account for sex differences observed in nerve regeneration and neuron loss following cranial nerve injury.

Fetal transplants rescue axial muscle representations in M1 cortex of neonatally transected rats that develop weight support.

Fetal transplants rescue axial muscle representations in M1 cortex of neonatally transected rats that develop weight support. J. Neurophysiol. 80: 3021-3030, 1998. Intraspinal transplants of fetal spinal tissue partly alleviate motor deficits caused by spinal cord injury. How transplants modify body representation and muscle recruitment by motor cortex is currently largely unknown. We compared electromyographic responses from motor cortex stimulation in normal adult rats, adult rats that received complete spinal cord transection at the T8-T10 segmental level as neonates (TX rats), and similarly transected rats receiving transplants of embryonic spinal cord (TP rats). Rats were also compared among treatments for level of weight support and motor performance. Sixty percent of TP rats showed unassisted weight-supported locomotion as adults, whereas approximately 30% of TX rats with no intervention showed unassisted weight-supported locomotion. In the weight-supporting animals we found that the transplants enabled motor responses to be evoked by microstimulation of areas of motor cortex that normally represent the lumbar axial muscles in rats. These same regions were silent in all TX rats with transections but no transplants, even those exhibiting locomotion with weight support. In weight-supporting TX rats low axial muscles could be recruited from the rostral cortical axial representation, which normally represents the neck and upper trunk. No operated animal, even those with well-integrated transplants and good weight-supported locomotion, had a hindlimb motor representation in cortex. The data demonstrate that spinal transplants allow the development of some functional interactions between areas of motor cortex and spinal cord that are not available to the rat lacking the intervention. The data also suggest that operated rats that achieve weight support may primarily use the axial muscles to steer the pelvis and hindlimbs indirectly rather than use explicit hindlimb control during weight-supported locomotion.

Increased concentrations of atrial and plasma atrial natriuretic peptide in castrated male rats

The effects of orchiectomy and testosterone replacement on the plasma concentration and the atrial stores of atrial natriuretic peptide (ANP) were studied in the rats. Male rats were orchiectomized (Orc) three weeks before replacement with testosterone propionate (TP, 20 mg/ml/kg body weight) or sesame oil for five days. Immunoreactive ANP (IR-ANP) in the extracted right atria and plasma of experimental rats was measured. Plasma ANP concentrations were 206 ± 22, 927 ± 151, and 264 ± 61 pg/ml in normal control, Orc, and Orc + TP rats, respectively. ANP contents in right atria were higher in Orc (108 ± 9 ng/mg tissue) and TP-treated Orc rats (123 ± 9 ng/mg tissue) than in normal animals (32 ± 7 ng/mg tissue). These results indicate an increased plasma concentration and atrial stores in the castrated male rats. Replacement of testosterone in the castrated male rats does not decrease the atrial ANP stores, but decreases the plasma ANP concentration.

Hypothalamic neurotransmitter function in experimentally induced hyperprolactinemia

It is known that animals or patients bearing a prolactin (PRL)-secreting tumor (PST) do not suppress PRL levels after administration of indirectly acting dopamine agonists, namely nomifensine (Nom), and are not responsive to the PRL releasing effect of antidopaminergic drugs and opioid peptides. Since the action of these drugs is mediated through the tuberoinfundibular dopaminergic (TIDA) system, these findings have been taken to indicate that animals and humans bearing prolactinomas have a defective TIDA function. Alternatively, PRL unresponsiveness to these drugs could be due to hyperfunction of TIDA system for the feedback action of high PRL levels. To clarify whether hypo- or hyperfunction of the TIDA system was responsible for such behaviour, we tested the effect of a synthetic opioid peptide (FK 33–824), a DA receptor antagonist, domperidone (Dom), and of Nom on PRL secretion in two experimental models of non-tumoral hyperprolactinemia, i.e. rats bearing ectopic pituitaries since 3 days (TP rats), or treated with ovine PRL (oPRL 250 μg, twice daily for 3 days), in which existence of an increased TIDA function has been demonstrated. FK 33–824 (0.5 mg/kg i.p.) increased significantly plasma PRL levels in control rats but failed to do so in TP rats and it elicited a significantly lower PRL response than in controls in rats treated with oPRL. In both experimental models, a PRL secretagogue, e.g. 5-hydroxytryptophan (50 mg/kg i.p.), elicited the same response as in controls, indicating that the pituitayr PRL pool was preserved. Kinetic characteristics of opioid binding sites in the hypothalamus of hyperprolactinemic rats did not differ from those of controls, and also hypothalamic Met-enkephalin concentrations were similar to those of controls. These findings would exclude a defect of endogenous opioid neurotransmission as responsible for the impaired PRL stimulation by the enkephalin analog. Supporting this were the findings that oPRL-treated rats exhibited a growth hormone response to FK 33–824 and a rise in plasma luteinizing hormone after naloxone (5 mg/kg s.c.) similar to those present in controls. These data indicate that the defective PRL responsiveness to opioids has to be attributed to the impairment of a neurotransmitter e.g. DA, functionally located at a site ‘downstream’ from opioid receptors. As already shown for oPRL-treated rats, TP rats exhibited a faster turnover rate of DA in the median eminence of the hypothalamus. In keeping with the idea that the hypothalamic dopaminergic system was involoved in the defective PRL responsiveness to opioids, Dom (50 μg/kg i.p.) induced aignificantly lower increase of PRL levels in TP and oPRL-injected rats than in controls. All these data demonstrate that opioid peptides and DA receptor antagonists are unable to discriminate the functional state of TIDA system. In fact, they fail to elicit a rise in plasma PRL in hyperprolactinemic states related to either TIDA hypo- (PST rats and humans) or hyperfunction (TP and oPRL-treated rats). In contrast to FK 33–824 and Dom, Nom (10 mg/kg i.p.), a drug which fails to lower PRL secretion in animals and patients with prolactinoma, did suppress plasma PRL levels of oPRL-treated rats as those of controls. The latter data indicate that indirectly acting DA agonists can discriminate the functional state of TIDA system; they fail to lower PRL secretion only when TIDA function is defective.

The effect of protein depletion upon tumor growth and host survival

Malnutrition has been implicated as an important factor in the host's response to tumor. This study examined the role of protein depletion upon tumor growth and host survival in 20 male Fischer F344 rats (250 g). Ten rats were inoculated subcutaneously with 5 × 10 6 tumor cells (methylcholanthrene-induced bladder carcinoma). For 14 days all 20 rats were maintained on regular diet pellets (23% protein) (P). By Day 8, the 10 inoculated rats had palpable tumors. On Day 14, half the tumor (T) and control (C) rats were placed on a protein-free (PF) diet. CP rats gained weight gradually reaching a plateau at 325 g by Day 60. TP rats maintained their body weights between 250 and 270 g. TPF and CPF rats lost about 45 g/rat by Day 28; TPF rats lost significantly more weight during the ensuing 36 days. TP and CP rats consumed 15–20 g diet/day while TPF and CPF ate 10–15 g. Tumor growth was similar up to Day 28 after which TP rats had significantly increased tumor volumes. By Day 57, 4 5 TP rats were dead with metastatic disease while only 1 5 TPF rats had died. Mean survival for the TPF rats was 3.8 days longer than that of TP rats. These results demonstrate that tumor-bearing rats on a 23% protein diet have larger tumors and do not survive longer than rats on a protein-free diet. This observation may have significant implications for the nutritional management of cancer patients.

Intestinal disaccharidases in newborn and mother rats: effect of dietary protein deficiency during pregnancy

Pups were delivered at term, by cesarean section, from control (CP) and test (TP) rats. The diets fed during pregnancy contained 26% casein (CP rats) or 6% casein (TP rats). At birth, body weight and small intestinal length and weight were significantly lower in pups of TP than in those of CP rats. Specific activity of lactase (µmoles of lactase hydrolyzed per minute per gram of protein) in the small intestine was similar in the two groups of pups (mean 106 versus 116). However, total activity of lactase in the entire small intestine was significantly lower in the pups of TP than in those of CP rats. In the mothers, the weight of the small intestine in TP rats was almost half that in the CP rats (3.7 versus 7.6 g). The atrophy was noted along the length of the intestine. In the mucosal scrapings of distal third of the small intestine, specific activities of the disaccharidases were significantly greater in TP than in CP rats (lactase, 15.0 versus 6.2; sucrase, 39 versus 21; maltase, 250 versus 152). In the proximal third of the small intestine, only maltase activity was significantly different (lactase, 12.0 versus 10.8; sucrase, 76 versus 61; maltase, 350 versus 258). Total activities of the disaccharidases in the small intestine (µmoles hydrolyzed per minute, by mucosal scrapings of the entire intestine) were significantly lower in the TP than CP rats (lactase, 4.0 versus 6.4; sucrase, 15 versus 28; maltase, 74 versus 125).

Strain differences for preoptic stimulation of ovulation in cyclic, spontaneously persistent-estrous, and androgen-sterilized rats.

The CD (Charles River) strain of rats differs in several important respects from our inbred Osborne Mendel (O-M) strain. The critical period for the ovulatory surge of gonadotropin in proestrus is less sharply limited in CD rats. They are much more refractory to preoptic (POA) electrochemical stimulation for ovulation, not only in normally cycling females, but strikingly so in androgen-sterilized (TP) females, as well as in rats having spontaneous persistent estrus (PE). In proestrous 4-day cyclic CD females ‘blocked’ with pentobarbital, a much larger stimulus (100 µA × 30 sec = 3000 µcoulombs) was required to fully ovulate all subjects than was needed in similar O-M rats (10 µA × 20 sec = 200 µcoulombs). While 5/6 O-M PE rats ovulated in response to massive unilateral POA stimulation (3000 µcoulombs), all of 6 CD PE rats failed to ovulate. Even bilateral POA stimulation with these parameters induced ovulation in only 3/10 rats. O-M TP rats had low POA thresholds close to those of cyclic O-M females, while only 1/6 CD TP rats gave even a partial response (5 tubal ova) to massive bilateral stimulation. CD TP rats also differed from O-M TP rats by secreting abundant prolactin in spite of the constant estrus. This was shown by prominent milk cysts in the mammary glands at 8–9 months of age and by spontaneous pseudopregnancy after corpora lutea were induced by LH injection. O-M TP rats had no mammary development and experienced only a short diestrus after LH injection. Whether there is a common basis for these strain differences remains uncertain.