Conditioned response to apomorphine in nigro-striatal system-lesioned rats: the origin of undrugged rotational response

Development and time-course characteristics of Early rotational response (ER) to apomorphine in 6-hydroxydopamine-lesioned rats is explored. We show here how this ER can be considered a conditioned response that arises when the drug is repeatedly administered, according to a classical conditioning paradigm. In this way, the ER to apomorphine can be considered a non-pharmacological, conditioned, placebo response, the drug action being the unconditioned stimulus (UCS). In our model, the undrugged rotational response elicited by saline injections two weeks after drug treatment can be considered as the conditioned response (CR) to the conditioned stimulus, the CS being the environment associated with the drug treatment. This CR had not previously been identified during the drug treatment. Thus, we studied the acquisition of the ER, nonexistent after the first injection of apomorphine. Furthermore, we distinguish between this ER and the later, strictly pharmacological rotational response (LR) to apomorphine. Finally, we related this ER to the undrugged, paradoxical response to saline. In conclusion, we demonstrate the paradigm of pharmacological conditioning using this animal model of Parkinson's disease, supported by our own results and those of Silverman and Ho (1981).     

Ethanol induces rotational behavior in 6-hydroxydopamine lesioned mice

Mice with unilateral striatal lesions created by 6-hydroxydopamine (6HDA) injection were screened for rotational (circling) behavior in response to injection of amphetamine and apomorphine. Those that rotated ipsilaterally in response to amphetamine and contralaterally in response to apomorphine were subsequently challenged with 1 to 3 g/kg (i.p.) ethanol. Surprisingly, ethanol induced dose related contralateral (apomorphine-like) rotation which, despite gross intoxication, was quite marked in most animals. No significant correlation was found between the number of turns made following ethanol and made after apomorphine or amphetamine.     

Circling behavior in old rats after unilateral intranigral injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Old and young adult rats received unilateral injections of MPTP or saline into the substantia nigra. Unilateral injection of MPTP in old rats induced ipsiversive circling on day 1 and day 7 after the injection; contraversive circling behavior was induced in MPTP-treated rats by systemic administration of apomorphine. Young rats showed ipsiversive circling on day 1 but not on day 7 after the injection; administration of apomorphine did not induce contraversive circling. On day 10 after the injection of MPTP, the concentration of D-2 receptors in the striatum of the injected hemisphere of old rats was increased by about 25% compared with the striatum of old rats with saline injection and of young rats with MPTP or saline injections. These results suggest that MPTP exerts neurotoxic effects on the nigrostriatal dopaminergic system of old rats and produces supersensitive dopamine receptors in the ipsilateral denervated striatum.     

Apomorphine induces latent rotation in lesioned rats, amphetamine does not

Rats with unilateral 6-hydroxydopamine (6HDA) lesions of one nigrostriatal pathway were used to compare the efficacy of apomorphine (Apo) and (+)-amphetamine (Amph) at inducing latent rotational behavior. Rats treated with Apo rotated contralaterally acutely after drug treatment and subsequently, weeks after drug treatment, exhibited rapid contralateral circling when re-introduced to the rotation environment. Amph treated animals, on the other hand, rotated ipsilaterally acutely after drug treatment and exhibited no latent drug effect.     

Early pituitary LH response to injections of GnRH in ewes

In the deep anoestrous period (June), five intact ewes and five ovariectomized ewes received 50 ug synthetic gonadotrophin-releasing hormone (GnRH). In the mid-breeding season (October), the GnRH administrations were repeated in five intact and four ovariectomized ewes; the former were in the luteal phase of the cycle. Blood samples were collected every 30 sec for 15 min, then at 15-min intervals. Release of luteinizing hormone (LH) occurred as soon as the second minute after injection in all ewes. This early response was earlier and more abrupt in the ovariectomized ewes than in the intact animals. In a second experiment three intact ewes that were in deep anoestrus received 50 ug GnRH followed 5 h 20 min later by a second identical injection. Another three intact ewes in deep anoestrus received two injections of 1 ug GnRH. Blood samples were taken every 15 sec for 15 min, then every 20 min until the next injection, and for a further 5 h after the second injection. This regimen was repeated in mid-breeding season during the luteal phase. There was again a very early release of LH; the magnitude of response was similar after the first injection of either 50 ug or 1 ug GnRH to intact ewes either in the breeding season or during deep anoestrus. However, a greater early release of LH was obtained at the lower dose only after the second injection of GnRH. Apart from this exception, the similar early release of LH occurred in spite of different amounts of LH released thereafter in response to the two doses of GnRH. It is suggested that the early response to GnRH consists of LH stored in a "readily releasable" pool in the pituitary, whereas the main release of LH may be a result of increased synthesis and/or release of a more stable pool.     

Effects of short- and long-acting dopamine agonists on sensitized dopaminergic neurotransmission in rats with unilateral 6-OHDA lesions

The effects of short and long-acting dopamine agonists on sensitized dopaminergic transmission in an animal model of Parkinson's disease were investigated. Rats with 6-hydroxydopamine (6-OHDA) lesions of the left nigrostriatal dopaminergic pathway were pre-exposed i.p. to 50 mg/kg methyl levodopa for 10 days. After a 7-day withdrawal period, these animals were treated with saline i.p., 0.05 mg/kg apomorphine s.c., or 0.5 mg/kg cabergoline i.p., once daily for 7 days. On the 8th day, rats in each treatment group received a challenge dose of 0.05 mg/kg apomorphine or saline s.c. The temporal changes in the number of rotations away from the 6-OHDA lesion side were evaluated after the challenge. The apomorphine challenge increased the number of rotations more markedly in the apomorphine pretreated rats than in the other pretreatment groups. In cabergoline pretreated rats, the number of rotations was significantly lower than that of saline-pretreated animals. Pretreatment with saline did not alter the apomorphine sensitivity of rotational behavior. These findings suggest that the repeated administration of long-acting dopamine agonists may reduce sensitized dopaminergic transmission in dopamine-depleted rats, whereas short-acting ones may further enhance sensitization of the transmission process.     

Development of tolerance in rats to the hypothermic effects of D-amphetamine and apomorphine

Rats given d-amphetamine (15 mg/kg i.p.) or apomorphine (10 mg/kg i.p.) and placed in a cold environment (4°C) developed marked hypothermia. After daily injections of either drug for seven weeks, the maximal hypothermic responses to d-amphetamine or apomorphine were reduced to 72% and 19% of those obtained initially. Subsequent injection of ET-495, a central dopamine receptor stimulant, caused rectal temperature to decrease only 72% and 49% as much as in control animals. The hypothermic response to apomorphine was also depressed in d-amphetamine-treated animals. These observations suggest that the tolerance to the hypothermic effects of both d-amphetamine and apomorphine that develops is due at least in part to alterations in the sensitivity of dopamine receptors.     

Decreased ability to cope with heat and cold linked to a dysfunction in a central dopaminergic pathway in elderly rats

Rectal and tail skin temperature was measured in lightly restrained rats aged 2 to 24 months after either drug injection or exposure to a hot or cold environment. At 18 months the rats were significantly less able to maintain their core temperature within the physiological range when subjected to a heat load, and this deficit was even more pronounced at 24 months, when an inability to maintain their core temperature in the cold was also evident. The hypothermic response of 24 month old rats to apomorphine and amphetamine was significantly less than that in 2 month old rats. In contrast, the elderly animals were more susceptible to the hypothermic effects of oxotremorine. These results suggest that a dysfunction in a dopaminergic pathway in the central nervous system can, at least in part, explain the inability of elderly rats to cope with heat stress.     

Selective interaction of drugs with a discriminable stimulus associated with narcotic actions

Rats were trained to bar press on either one of two levers depending on whether they received an injection of morphine (10 mg/kg) or saline. The rats responded on the morphine-correct lever when injected with another narcotic, fentanyl, but responded on the saline-correct lever when injected with a narcotic antagonist or another CNS active, but non-narcotic, drug (e.g., amphetamine, apomorphine). The narcotic antagonist, naloxone, prevented the occurrence of the narcotic discriminable stimulus, but the rats responded on the morphine-correct lever when injected with morphine plus any of a number of potent CNS active, but non-narcotic compounds. These results are discussed with reference to the specificity of this procedure for detecting drugs with narcotic agonist or antagonist properties.     

Increased ability of apomorphine to reduce serum concentrations of prolactin in rats treated chronically with alpha-methyltyrosine

Serum concentrations of prolactin were significantly increased in rats for up to 9 hours after a single i.p. injection of α-methyltyrosine (50 mg/kg); apomorphine caused a dose-dependent reduction of the elevated prolactin concentrations. Doses of apomorphine required to reduce serum prolactin concentrations.were lower in animals previously injected with α-methyltyrosine three times a day for 10 days than in animals which received a single injection of α-methyltyrosine. These results suggest that chronic disruption of the normal release of dopamine from tuberoinfundibular neurons leads to the development of increased sensitivity of dopamine receptors involved with the inhibition of prolactin release from the anterior pituitary.     

5-HT1A receptor agonist 8-OH-DPAT acts in the hindbrain to reverse the sympatholytic response to severe hemorrhage

Central administration of serotonergic 5-HT1A receptor agonists delays the reflex sympatholytic response to severe hemorrhage in conscious rats. To determine the region where 5-HT1A receptor agonists act to mediate this response, recovery of mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) was compared in hemorrhaged rats after injection of the selective 5-HT1A agonist, (+)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), in various regions of the cerebroventricular system or the systemic circulation. Three minutes after injection of 8-OH-DPAT (48 nmol/kg), MAP and RSNA were higher in hemorrhaged rats given drug in the fourth ventricle (94 +/- 5 mmHg, 82 +/- 18% of baseline) or the systemic circulation (90 +/- 4 mmHg, 113 +/- 15% of baseline) than in rats given drug in the Aqueduct of Sylvius (63 +/- 4 mmHg, 27 +/- 11% of baseline), the lateral ventricle (42 +/- 3 mmHg, -8 +/- 18% of baseline), or in rats given saline in various brain regions (47 +/- 5 mmHg, -42 +/- 10% of baseline). A lower-dose injection of 8-OH-DPAT (10 nmol/kg) also accelerated the recovery of MAP and RSNA in hemorrhaged rats when given in the fourth ventricle (94 +/- 26 mmHg, 72 +/- 33% of baseline 3 min after injection) but not the systemic circulation (46 +/- 4 mmHg, -25 +/- 30% of baseline). These data indicate that 8-OH-DPAT acts on receptors in the hindbrain to reverse the sympatholytic response to hemorrhage in conscious rats.     

Pituitary response of prepuberal lambs to oestradiol-17 beta.

In two experiments 48 prepuberal Merino ewe lambs were injected with oestradiol-17 beta (E2) or saline to study the effect of E2 on their plasma LH levels and on oestrus and ovulation. In the three groups which received 30 (experiment I), 50 and 30 (experiment II) microgram E2 respectively, 27 out of 28 lambs showed an LH response, the corresponding mean LH peaks being 64.3 +/0 22.5, 153.6 +/-33.4 and 91.7 +/- 16.9 ng/ml at mean intervals of 11.1, 11.2 and 10.5 h, respectively, after injection. None of the 20 lambs in the control groups had an LH level higher than 18 ng/ml 12 h after injection. In the three E2 groups, 41.7, 62.5 and 37.5% of animals showed oestrus within 26 h of injection while in the control groups only one animal showed oestrus. Of 13 animals showing oestrus in the E2 groups, 11 failed to ovulate. The mean pre-injection plasma FSH level in experiment I was 102.7 ng/ml, and in four 5--7-month-old lambs over several weeks uas 155.3 ng/ml. Despite these high pre-injection levels of FSH, it appears that the follicles were unable to respond to the LH peak which followed the E2 injection.     

Fast desensitization of the response to InsP3 in Limulus ventral photoreceptors.

In Limulus ventral photoreceptor cells the time-course of the desensitization of InsP3 response was measured by an injection-pair paradigm. Pressure pulses of InsP3 were delivered into the cell with various interpulse intervals. The desensitization of the response to the second injection of each pair approached totality at 200 ms, which is the duration of the response to a single pressure pulse of InsP3. Lowering extracellular calcium did not affect the time-course of the desensitization. Lowering the temperature slowed down both the time-course of the response to InsP3 and the time-course of the desensitization to the same extent. These findings suggest that the desensitization is powerful enough and its onset fast enough to contribute to the transience of the InsP3 response. The time-course of the desensitization suggests it may influence light adaptation.     

Regenerative Therapeutic Potential of Adipose Stromal Cells in Early Stage Diabetic Retinopathy

Diabetic retinopathy (DR) is the leading cause of blindness in working-age adults. Early stage DR involves inflammation, vascular leakage, apoptosis of vascular cells and neurodegeneration. In this study, we hypothesized that cells derived from the stromal fraction of adipose tissue (ASC) could therapeutically rescue early stage DR features. Streptozotocin (STZ) induced diabetic athymic nude rats received single intravitreal injection of human ASC into one eye and saline into the other eye. Two months post onset of diabetes, administration of ASC significantly improved “b” wave amplitude (as measured by electroretinogram) within 1–3 weeks of injection compared to saline treated diabetic eyes. Subsequently, retinal histopathological evaluation revealed a significant decrease in vascular leakage and apoptotic cells around the retinal vessels in the diabetic eyes that received ASC compared to the eyes that received saline injection. In addition, molecular analyses have shown down-regulation in inflammatory gene expression in diabetic retina that received ASC compared to eyes that received saline. Interestingly, ASC were found to be localized near retinal vessels at higher densities than seen in age matched non-diabetic retina that received ASC. In vitro, ASC displayed sustained proliferation and decreased apoptosis under hyperglycemic stress. In addition, ASC in co-culture with retinal endothelial cells enhance endothelial survival and collaborate to form vascular networks. Taken together, our findings suggest that ASC are able to rescue the neural retina from hyperglycemia-induced degeneration, resulting in importantly improved visual function. Our pre-clinical studies support the translational development of adipose stem cell-based therapy for DR to address both retinal capillary and neurodegeneration.     

An experimental model of tardive dyskinesias

Rats treated continually and chronically with trifluoperazine (ca 3 mg/kg/day) for six months initially developed mild catalepsy and an inhibition of spontaneous locomotor activity; both effects disappeared by three months. An initial increase in dopamine turnover (as measured by levels of homovanillic acid and dihydroxyphenylacetic acid) also disappeared by three months. Apomorphine-induced stereotypy was completely inhibited in drug-treated animals at two weeks, but progressively returned to normal after three months of drug intake. An exaggerated response to apomorphine developed in animals after six months of drug administration. Inhibition of striatal dopamine-stimulated adenylate cyclase found during the first month of drug intake was reversed at three months, a trend exaggerated after continuous drug administration for six months. Specific striatal ³H-spiperone binding affinity decreased acutely, but was increased after six months drug intake; no change in number of receptor sites occurred.These changes suggest that at least striatal dopamine receptors may become “supersensitive” during chronic neuroleptic treatment.     

Immune response to a hepatitis B DNA vaccine in Aotus monkeys: a comparison of vaccine formulation, route, and method of administration.

BACKGROUND: Attempts to optimize DNA vaccines in mice include using different routes of administration and different formulations. It may be more relevant to human use to carry such studies out in nonhuman primates. Here we compare different approaches to delivery of a DNA vaccine against the hepatitis B virus (HBV) in Aotus monkeys. MATERIALS AND METHODS: Thirty-two adult Aotus l. lemurinus monkeys divided into 8 groups of four were immunized with 400 microg of a DNA vaccine which encoded hepatitis B surface antigen (HBsAg). DNA in saline was administered by intradermal (ID) or intramuscular (IM) injection with needle and syringe, IM injection with the Biojector needleless injection system or combined ID (needle) and IM (Biojector). DNA formulated with cationic liposomes (CellFECTIN) was injected IM with needle or Biojector. DNA with added E. coli DNA (100 microg) was injected IM with the Biojector or ID. A ninth group of 4 monkeys was injected IM (needle) with Engerix-B, a commercial vaccine containing recombinant HBsAg (10 microg) adsorbed onto alum. Monkeys were boosted in an identical fashion to their prime at 8 weeks, but all received the protein vaccine (Engerix-B) at 16 weeks. Sera was assessed for antibodies against HBsAg (anti-HBs) by enzyme-linked imunosorbent assay (ELISA). RESULTS: The primary humoral response induced by IM delivery of the DNA vaccine was very poor. In most cases there was no detectable anti-HBs even after 2 DNA doses but the kinetics of the response to subsequent protein indicated that a memory B cell response had been induced. In contrast, following IM-administration of DNA using the Biojector, detectable anti-HBs were observed in 3 of 8 animals and evidence for immunological priming was apparent in an additional 4 of the 8 monkeys. ID injection of DNA vaccine in saline induced a potent antibody response which was augmented 6-fold by the addition of E. coli DNA. Combining ID and IM administration did not improve humoral immunity over ID injection alone. CONCLUSIONS: For immunization of primates with DNA vaccines, ID may be a preferable route to IM, although it is not clear whether the Aotus monkey is a relevant model for humans in this respect. Nevertheless, the use of the Biojector needleless injection system may improve responses with IM delivery of DNA vaccines. As well, the immunostimulatory action of E. coli DNA may be used to augment the humoral response induced by a DNA vaccine.     

Protective effects of N-acetylserotonin against 6-hydroxydopamine-induced neurotoxicity

The present work studied in vivo neuroprotective effects of n-acetylserotonin (NAS), the immediate precursor of melatonin, on the dopaminergic system, in rats lesioned with the unilateral intrastriatal injection of the neurotoxin 6-hydroxydopamine (6-OHDA). Two weeks after the lesion, the dopamine receptor agonist, apomorphine, produced rotational asymmetry, and the NAS treatment significantly reduced the motor deficit following the apomorphine challenge. The apomorphine-induced rotational behavior was blocked by 84, 86 and 53% after NAS, at doses of 2, 5 and 10 mg/kg, i.p., respectively. The injection of 6-OHDA significantly decreased DA, DOPAC and HVA levels in the rat striatum. In contrast, the NAS (2, 5 and 10 mg/kg, i.p., daily for 7 days) treatment partially reversed the decreases caused by 6-OHDA, and the neurotransmitter levels were brought to approximately 50% of that observed in the contralateral sides. NAS was more efficient at the smaller doses. NAS (5 mg/kg) produced an up-regulation of D1 (37%) and D2 (37%) receptors associated with a decrease in Kd values.     

Time course analyses of the thermoregulatory responses to melatonin and chlorpromazine in bull snakes (Pituophis melanoleucus)

Intraperitoneal injections of either melatonin (MEL) or chlorpromazine (CPZ) significantly lowered preferred body temperature (T_b) of bullsnakes, Pituophis melanoleucus. Multiple comparison procedures showed that T_b for both MEL and CPZ treatments differed significantly from both the injected and non-injected control groups. However, T_b for MEL treatments did not differ from those of CPZ treatments (t=0.471; df=12; P=0.646), indicating that each drug has a similar effect on thermal selection. Our results obtained from time-course experiments support earlier experiments where a repeated measures design was used to test treatment effects of MEL and CPZ on thermal selection. However, we further show that injected controls (saline and ethanol injection) do not differ from non-injected controls (no injection) for both MEL and CPZ experiments. Although there are no differences between T_b of snakes receiving MEL or CPZ, time-course analyses indicate that the duration of thermoregulatory responses differ between MEL and CPZ treatments. This indicates that CPZ treatments may be more effective in increasing the amount of time required for individuals to return to normal preferred T_b or set temperature (T_set). The initial duration of thermoregulatory responses to the first injection of MEL differed significantly from the second injection. There were no differences in the duration of thermoregulatory responses between the first and second injections of CPZ. There are no data for the metabolic half-life of MEL in ectothermic vertebrates. Our study provides some information regarding the time-course of thermoregulatory responses to elevated levels of MEL via intraperitoneal injections of either MEL or CPZ, a metabolic antagonist of MEL.     

Pineal N-acetyltransferase, pineal and serum melatonin response to isoproterenol stimulation in underfed male rats

Adult male rats were subjected to 4 weeks of 50% food restriction under lighting regimen of 14 h light and 10 h dark. The pineal response to isoproterenol (ISO) was determined. In the time-course study, animals were injected with 0.5 mg/Kg ISO subcutaneously (SC) and killed at different times up to 180 min post injection. In the dose-response study, various doses of ISO (0.2 mg/Kg to 5.0 mg/Kg) were injected intraperitoneally (IP) and animals were killed 120 min post injection. Body weight, pineal N-acetyltransferase (NATase), pineal and serum melatonin (MT) were determined. After 4 weeks of restricted feeding, body weight was reduced by 40%. In the time-course study, peak pineal NATase occurred 120 min post injection in the ad libitum fed animals. By contrast, the food restricted animals showed a gradual increase of pineal NATase up to 180 min post injection. In the dose-response study, the ad libitum fed animals demonstrated a dose dependent increase of pineal NATase up to 5 mg/kg dose. The food restricted animals, however, achieved their maximal pineal NATase at 1 mg/Kg dose with no further increment at 5 mg/Kg dose. These differences in responsiveness were also reflected in pineal and serum MT levels. These results indicate that underfed animals have abnormal pineal NATase, pineal and serum MT responses to ISO stimulation.     

Safety of photosynthetic Synechococcus elongatus for in vivo cyanobacteria–mammalian symbiotic therapeutics

The cyanobacterium Synechococcus elongatus (SE) has been shown to rescue ischaemic heart muscle after myocardial infarction by photosynthetic oxygen production. Here, we investigated SE toxicity and hypothesized that systemic SE exposure does not elicit a significant immune response in rats. Wistar rats intravenously received SE (n = 12), sterile saline (n = 12) or E. coli lipopolysaccharide (LPS, n = 4), and a subset (8 SE, 8 saline) received a repeat injection 4 weeks later. At baseline, 4 h, 24 h, 48 h, 8 days and 4 weeks after injection, clinical assessments, blood cultures, blood counts, lymphocyte phenotypes, liver function tests, proinflammatory cytokines and immunoglobulins were assessed. Across all metrics, SE rats responded comparably to saline controls, displaying no clinically significant immune response. As expected, LPS rats exhibited severe immunological responses. Systemic SE administration does not induce sepsis or toxicity in rats, thereby supporting the safety of cyanobacteria–mammalian symbiotic therapeutics using this organism.