Time-course analysis and comparison of acute and chronic intrastriatal quinolinic acid administration on forelimb reaching deficits in the rat.

Rats were trained to use a single forelimb for a food pellet retrieval task. During baseline testing all rats exhibited > 90% use of a preferred limb for the task. Following baseline, rats were subjected to chronic administration (18 day) or acute injection of quinolinic acid (QUIN) or vehicle to the striatum contralateral to the preferred limb. Rats were tested 48 h after insertion of chronic delivery probes or after acute injection and retested every 48 h over an 18-day period. Compared to vehicle, rats receiving chronic QUIN (7.6 nmol/h) exhibited an increase in the number of reach attempts required to meet task criteria. Chronic QUIN did not produce a significant change in latency to initiate the task or an increase in latency to complete the task. No rats exposed to chronic QUIN exhibited a switch in limb preference for the task. Unlike animals exposed to chronic QUIN, a significant number of animals receiving acute QUIN injections switched to exclusive use of the ipsilateral (nonpreferred) limb for the task. Quantitative histological analysis revealed no significant difference in lesion volume between acute and chronic lesion animals. These findings suggest that behavioral manifestations of histopathologically similar lesions may be vastly different depending on the methods used to produce these lesions. More specifically, the acute injection model resulted primarily in forelimb disuse, whereas the chronic model resulted in continued abnormal use of the affected limb. Understanding adaptive strategies used in these models may be particularly important when testing newly developed transgenic models of neurodegenerative diseases and the therapeutic potential of newly developed neuroprotectants.

Chronic administration of quinolinic acid in the rat striatum causes spatial learning deficits in a radial arm water maze task.

Chronic intrastriatal administration of quinolinic acid (QA) in the rat produces a pattern of neurodegeneration similar to that seen in Huntington's disease (HD). Although these changes have been related to transient motor abnormalities, the effects of chronic QA administration on cognitive abilities have not been assessed. The present study investigated whether the striatal deterioration observed during chronic QA administration produces cognitive impairments in this animal model of HD by testing the effects of chronic administration of QA on spatial learning ability of rats in a radial arm water maze (RAWM) task. Rats were given bilateral implantation of a chronic dialysis probe apparatus which delivered either vehicle or QA (20 mM) into the striatum. Beginning 1 day after implantation, the rats were tested daily for 3 weeks in the RAWM. Nocturnal activity levels were also assessed at 1-, 3-, 5-, 7-, 14-, and 21-days following probe implantation. Results of behavioral testing indicated that chronic exposure to QA causes spatial learning deficits in the RAWM task with only a transient increase in activity levels. Collectively, these results suggest that chronic striatal exposure to QA mimics some aspects of the cognitive deficits observed in HD.

The neuronal NOS inhibitor L-MIN, but not 7-NINA, reduces neurotoxic effects of chronic intrastriatal administration of quinolinic acid.

Rat striata were exposed to 15 mM quinolinic acid (QUIN), or QUIN plus the nitric oxide synthase inhibitors S-methyl-L-thiocitrulline dihydrochloride (L-MIN) or 7-nitroindazole monosodium salt (7-NINA) for 21 days. Co-administration of 100 microM or 1 mM L-MIN with QUIN significantly reduced lesion volume compared to QUIN alone. Co-administration of 1 microM or 10 microM L-MIN with QUIN had no significant effect. There was no significant effect of 7-NINA co-administered with QUIN compared to QUIN alone. L-MIN reduction of lesion volume supports the contention that neuronal nitric oxide synthase is a mediator of excitotoxic injury.

Synergistic effects of chronic exposure to subthreshold concentrations of quinolinic acid and malonate in the rat striatum.

Adult rats received chronic intrastriatal dialytic exposure to quinolinic acid (QUIN), malonate, or a combination of QUIN and malonate. The combination of subthreshold concentrations of QUIN (4 mM) and malonate (400 mM) produced lesions larger than did either QUIN or malonate alone. The neurotoxic effect of QUIN combined with malonate was subsequently blocked by co-administration of the NMDA receptor antagonist MK-801 (1 mM). These findings indicate that malonate synergistically enhances NMDA receptor mediated excitotoxicity.

Chronic intrastriatal administration of quinolinic acid produces transient nocturnal hypermotility in the rat.

Adult male Sprague-Dawley rats were exposed to 15 mM quinolinic acid solution or vehicle via bilateral intrastriatal dialytic administration for a period of 3 weeks. Animals were tested twice weekly for spontaneous behaviors and nocturnal activity during the 3-week dialysis period and for the 3 weeks following cessation of the dialysis period treatment. Nocturnal activity increased significantly (p < 0.005) during the first week of quinolinic acid exposure compared to vehicle exposed animals. The increase in nocturnal activity subsequently diminished to near control levels by the end of the 3-week dialysis period. During the 3-week period following cessation of dialysis, no significant differences were seen between quinolinic acid and vehicle-exposed animals. In addition, no differences were noted between quinolinic acid and vehicle-exposed animals in spontaneous behaviors either in the 3-week dialysis period or the 3-week period following cessation of dialysis. The results of this study are in agreement with other recent findings of transient nocturnal hyperactivity following striatal damage in rats. One possible explanation for the transient nature of this behavioral change is a transient effect of excitotoxicity in the striatum. During initial exposure to excitotoxins, nocturnal hypermotility could result from premorbid changes in neural function. With continued exposure, this behavioral effect may then diminish as a result of subsequent widespread striatal cell death.

Chronic administration of malonic acid produces selective neural degeneration and transient changes in calbindin immunoreactivity in rat striatum.

Adult rats received chronic dialytic delivery devices that exposed the striatum to a 100 mM, 400 mM, or 4 M solution of the reversible succinate dehydrogenase inhibitor malonic acid (MA). Three weeks of exposure to 100 or 400 mM MA produced no significant reduction in striatal cytochrome oxidase staining, whereas striata chronically exposed to 1 and 4 M MA showed a significant and dose-related reduction in cytochrome oxidase staining. In striata exposed to 1 M MA, analysis of regions radial to the necrotic core revealed significant reduction of nissl cell staining with relative sparing of NADPH-diaphorase-containing neurons. Although 100 and 400 mM MA failed to produce lesions, both of these concentrations significantly decreased the number of striatal calbindin (CALB) immunoreactive perikarya. The reduction in CALB immunoreactivity was partly reversed in animals allowed to survive 4 weeks after cessation of exposure to 400 mM MA. These results indicate that, like striatal lesions produced by quinolinic acid, lesions produced by chronic exposure to MA possess a Huntington's disease-like pattern of selective neurodegeneration. In addition, exposure to subthreshold MA concentrations (100 and 400 mM) produce widespread transient changes in striatal CALB that may be associated with a premorbid state of neuronal dysfunction.

Synergistic effect of intrastriatal co-administration of L-NAME and quinolinic acid.

Chronic dialytic intrastriatal co-administration of quinolinic acid (QUIN) and four concentrations of the nitric oxide synthase (NOS) inhibitor NG nitro-L-arginine methyl ester (L-NAME) produced variable results. Low concentrations of L-NAME (1 microM and 50 microM) co-administered with 15 mM QUIN produced lesions not significantly different from those produced by 15 mM QUIN alone. In contrast, higher concentrations of L-NAME (1 mM and 100 mM) co-administered with 15 mM QUIN produced striatal lesions significantly larger than those produced by 15 mM QUIN alone. Administered by itself, 100 mM L-NAME produced little striatal damage. These findings suggest that low levels of NOS inhibition have little or no effect on NMDA neurotoxicity in the striatum, whereas high levels of NOS inhibition increase NMDA-induced striatal lesion volume.

Chronic intrastriatal quinolinic acid produces reversible changes in perikaryal calbindin and parvalbumin immunoreactivity.

We recently reported the use of a chronic dialytic delivery system for intrastriatal administration of quinolinic acid in the rat. This system produces neurodegeneration with some characteristics similar to post mortem brain tissue from Huntington's disease patients, including reduced cytochrome oxidase staining, a decreased number of Nissl-stained neurons, and relative sparing of striatal NADPH-diaphorase containing neurons. The present findings show that chronic dialytic delivery of quinolinic acid also produces a Huntington's disease-like pattern of reduced calbindin and parvalbumin perikaryal immunoreactivity that is reversed in rats allowed four to eight weeks' recovery after cessation of quinolinic acid. Furthermore, cytochrome oxidase staining and the number of Nissl-stained cells were unchanged in the region of transient calbindin and parvalbumin immunoreactive perikaryal staining alterations. These results suggest that changes in calbindin and parvalbumin perikaryal immunoreactivity provide a relatively sensitive measure of quinolinic acid induced neurotoxicity. The reversible nature of reduced perikaryal immunoreactivity suggests a premorbid state of neurotoxicity, possibly marked by cellular redistribution of calbindin and parvalbumin.

Sex differences in the rapid and acute effects of estrogen on striatal D2 dopamine receptor binding.

Regional changes in striatal D2 dopamine (DA) receptor binding in castrated (CAST) or ovariectomized (OVX) rats were investigated following administration of a low dose of estradiol benzoate (EB), repeated treatment with EB followed by progesterone, or vehicle. In two separate experiments, there was a significant decrease in striatal D2 DA receptor binding in caudal striatum from OVX, but not CAST rats 30 min after a single injection of EB. In CAST rats, there was a significant increase in striatal D2 DA receptor binding in rostral striatum 4 h after injection of EB. There was no effect of EB plus progesterone treatment in either OVX or CAST rats. In addition, CAST rats had significantly lower D2 DA receptor binding in the lateral region of the rostral striatum than did OVX rats. These results show sexually dimorphic and regionally specific effects of estrogen on striatal D2 DA receptor binding, and a significant sex difference in striatal D2 DA receptor binding in the absence of circulating gonadal hormones. The present findings are discussed in light of previous reports of sex differences in gonadal hormone influences on striatal DA mediated behaviors.

Chronic intrastriatal dialytic administration of quinolinic acid produces selective neural degeneration.

The excitotoxic hypothesis of Huntington's disease pathogenesis suggests that selective striatal neuronal loss results from excessive activation of striatal excitatory amino acid receptors. Using a microdialysis probe mated to an Alzet 2002 mini-osmotic pump three different concentrations of quinolinic acid or vehicle were administered to the striata of rats over a 3-week period. Animals that received a total of 3.3 mumol of quinolinic acid had significant striatal atrophy that could be attributed to two distinct areas of neuronal loss. First, an area of necrosis surrounding the probe was marked by inflammatory infiltrate and a lack of neurons. In the second region, surrounding the necrotic area, there was a significant reduction in nissl-stained cells, with relative sparing of NADPH-diaphorase-staining neurons. In addition, there was a reduction in cytochrome oxidase staining throughout both of the areas of cell loss. Beyond the area of cell loss, the striatum appeared normal in all respects. The striata of animals that received 880 nmol quinolinic acid appeared identical to those that received vehicle. The striata of animals that received 8.8 mumol quinolinic acid showed severe nonselective atrophy of the striatum and some surrounding structures. We conclude that dialytic delivery of 3.3 mumol quinolinic acid produces an area of neuronal destruction that resembles the selective neuronal loss seen in Huntington's disease. This selective neurodegeneration produced by chronic exposure to quinolinic acid simulates more closely the course of Huntington's disease than previously described methods.

A new, simple myelin stain.

A new and convenient myelin stain is described. Paraformaldehyde fixed tissue is serially immersed in a nitroblue tetrazolium solution and then in a diaminobenzidine solution. The result is distinct blue staining of myelinated fiber tracts. This technique has advantages over presently used myelin stains.

Male rat copulation following 6-OHDA lesions of the medial preoptic area: resistance to repeated administration and rapid behavioral recovery.

Dopamine (DA) in the medial preoptic area (MPOA) has been shown to facilitate male rat sexual behavior. However, injections of the catecholamine (CA) neurotoxin 6-OHDA into the MPOA did not impair copulation in tests 3 days after injection. In the present study, three weekly (serial) injections produced no copulatory deficits compared to animals that received a single injection or to preinjection copulatory behavior scores. However, blocking CA synthesis, which did not impair control rats, produced deficits in both single and serial lesion animals, with significantly fewer serial than single lesion animals initiating copulation. Biochemical analysis of tissue punches showed no difference in MPOA concentrations of dopamine, norepinephrine, epinephrine, or the dopamine metabolite DOPAC between the two groups. Additional animals were tested at earlier intervals after 6-OHDA injections into the MPOA. Tests conducted 30 min after an MPOA injection of 6-OHDA revealed that all measures of copulation were impaired, relative to scores 24 h later. However, these scores were not significantly different from animals tested 30 min after a vehicle injection. A final group, tested 4 h after injection, showed impairment of all measures of copulation compared to vehicle injections and to tests 24 h later. Furthermore, in the tests 24 h later, 6-OHDA animals were not different from vehicle animals. Results from all experiments show that 6-OHDA injections into the MPOA impair copulation for at least 4 h, but that behavioral recovery is complete 24 h later. However, deficits can be reinstated by inhibiting DA synthesis, suggesting that increased synthesis in undamaged terminals contributed to behavioral recovery.

A novel device for chronic intracranial drug delivery via microdialysis.

A system is described for chronic intracranial drug administration in the rat using a modified in vivo microdialysis probe coupled to an Alzet model 2002 osmotic minipump. The results presented demonstrate that this system can be used for the chronic administration of quinolinic acid with minimal non-specific damage. Each pump delivered approximately 225 microliters of solution over a period of 19-20 days when tested in vitro. The dialysis units were uniform in function, delivering greater than 93% of the [3H]quinolinic acid initially loaded into the minipump. For in vivo analysis of this apparatus the dose of quinolinic acid tested produced extensive destruction of the striatum. The present system allows reliable drug diffusion over a relatively large area without pressure injection variability. In conclusion, we have developed a simple and inexpensive technique for administration of drugs into brain parenchyma with substantial advantages over previously used techniques.

Microinjection of the dopamine antagonist cis-flupenthixol into the MPOA impairs copulation, penile reflexes and sexual motivation in male rats.

Microinjection of the dopamine antagonist cis-flupenthixol into the medial preoptic area was previously shown to impair male rat copulatory behavior. The present experiments provide further evidence of cis-flupenthixol's inhibitory effects on male sexual behavior. Following microinjections of moderate to high doses of cis-flupenthixol, males exhibited slower copulatory rates and fewer ejaculations in copula, fewer ex copula erections and penile movements, and reduced sexual motivation in an X-maze. Locomotion in the X-maze was not significantly affected. Microinjections of the inactive isomer trans-flupenthixol produced no change in any behavioral measure, indicating that cis-flupenthixol's effects were receptor mediated. We suggest that dopamine receptors in the MPOA influence copulation primarily by regulating reflexive and motivational factors, but not locomotion.

Dose dependent D2 effects on genital reflexes after MPOA injections of quinelorane and apomorphine.

This study investigated the effects on genital reflexes of unilateral MPOA injections of 0.1, 1, 3, and 10 micrograms of the D2 agonist quinelorane (LY-163502), and of 3 micrograms quinelorane administered together with 3 micrograms of the D1 antagonist SCH-23390. In addition, the effects of an MPOA injection of 10 micrograms apomorphine were tested. All but the lowest dose of quinelorane significantly decreased the latency to the first reflex. The 3 and 10 micrograms doses of quinelorane, and the combination of quinelorane and SCH-23390, decreased the total number of reflexes. In addition, 10 micrograms quinelorane increased the number of seminal emissions. 10 micrograms apomorphine, like 10 micrograms quinelorane, decreased the latency to the first reflex and increased the number of seminal emissions, but did not decrease the numbers of erections or penile movements. The ratio of D1/D2 activity may influence the number of erections displayed during ex copula testing.

Dopamine receptors in the ventral tegmental area modulate male sexual behavior in rats.

The mesocorticolimbic dopamine tract is considered to be a substrate for motivation and reward as well as for locomotor behavior. The present experiments assessed the role of dopamine cell bodies in the ventral tegmental area (VTA), the source of this tract, in the copulatory behavior of male rats. The dopamine agonist apomorphine or the dopamine antagonist cis-flupenthixol were microinjected into the VTA immediately before sexual behavior tests with a receptive female. Apomorphine delayed the onset of copulation and slowed its rate, presumably by stimulating somatodendritic autoreceptors and thereby decreasing the firing rate of VTA neurons. Control injections of apomorphine into the substantia nigra were without effect. cis-Flupenthixol, which would have blocked autoreceptors and thereby depolarized VTA neurons, shortened the latency to begin copulating in those animals that did copulate; however, fewer animals exhibited sexual behavior. One possible explanation for the apparently contradictory effects of cis-flupenthixol may be that VTA neurons increased their rate of firing in some animals, leading to a faster onset of copulation, but that in other animals depolarization block in a substantial number of neurons resulted in a lack of copulation. These results are consistent with a contribution of the mesocorticolimbic dopamine tract to motivational and/or motor aspects of male copulatory behavior.

D2/D1 ratio in the medial preoptic area affects copulation of male rats.

The D1/D2 dopamine agonist apomorphine, microinjected into the medial preoptic area (MPOA), facilitates male rat sexual behavior and the D1/D2 antagonist cis-flupenthixol in the MPOA impairs it. The present study investigated the roles of D1 and D2 receptors in the regulation of copulation by microinjecting drugs selective for these receptors into the MPOA. The D2 agonist LY-163502 delayed the onset and slowed the rate of copulation and also reduced the number of vaginal intromissions required to trigger ejaculation (reduced ejaculatory threshold). The D1 agonist SKF-82526 had no effect, either alone or together with LY-163502. The D1 antagonist SCH-23390 delayed the onset of copulation and decreased ejaculatory threshold, as had the D2 agonist. A low dose of the D2 agonist alone and together with the D1 antagonist delayed the onset of copulation and reduced ejaculatory threshold; the combination of drugs was more effective than LY-163502 alone. Only the combination of drugs slowed the rate of copulation and delayed the resumption of copulation after an ejaculation. Thus, increasing the D2/D1 ratio in the MPOA, by selective stimulation of D2 and/or antagonism of D1 receptors, delays the onset of copulation and reduces ejaculatory threshold, possibly by altering autonomic control of penile reflexes.

Brain localization of cholinergic influence on male sex behavior in rats: antagonists.

The muscarinic receptor antagonist scopolamine was microinjected into either the preoptic area or the lateral ventricle, preceding sexual behavior tests. In Experiment 1 unilateral ventricular injections of scopolamine delayed the initiation of copulation, while unilateral preoptic injections had no effect. In Experiment 2 bilateral injections into the preoptic area produced dose-related decreases in the percentages of animals intromitting and ejaculating. In Experiment 3 scopolamine, injected alone into the preoptic area, again decreased the percentages of animals mounting, intromitting, and ejaculating. The muscarinic agonist oxotremorine, injected alone into the preoptic area, decreased ejaculatory threshold (i.e., decreased the number of intromissions preceding ejaculation) as previously reported. Concurrent oxotremorine and scopolamine injections into the preoptic area were not different from vehicle; thus, scopolamine blocked oxotremorine's effect. These data suggest that some cholinergic activation of the preoptic area is critical for normal copulation, since bilateral blockade of muscarinic receptors there dramatically decreased the number of animals copulating. However, increased cholinergic activity there only reduced ejaculation threshold.

Apomorphine and haloperidol, but not domperidone, affect penile reflexes in rats.

Previous studies have shown that systemic administration of the dopamine agonist apomorphine produced a biphasic effect on erection in the freely moving rat, with lower doses facilitating, and high doses inhibiting, erection. However, those studies did not distinguish between erection per se and seminal emission. The present results demonstrate that apomorphine produces a similar biphasic effect on penile reflexes in the restrained, supine rat, while facilitating seminal emission in a monophasic fashion. Haloperidol, a centrally-acting dopamine antagonist, either blocked the effects produced by apomorphine administration, or had actions opposite to those of apomorphine. Domperidone, a dopamine antagonist that does not readily penetrate the blood-brain barrier, did not antagonize apomorphine's effects, and did not affect penile responses when administered alone. These results suggest that dopamine receptors in the central nervous system regulate genital responses, and that effects on penile reflexes and seminal emission can be experimentally dissociated.

Microinjection of cis-flupenthixol, a dopamine antagonist, into the medial preoptic area impairs sexual behavior of male rats.

Systemically administered dopamine agonists have been shown to facilitate copulation in male rats. Microinjection of the dopamine agonist apomorphine into the medial preoptic area has also been reported to facilitate sexual behavior. The present experiments investigated the effects of medial preoptic microinjections of the dopamine antagonist cis-flupenthixol on male rat copulatory behavior. Fewer males initiated copulation and fewer ejaculated following flupenthixol administration. Those males that did ejaculate following flupenthixol injections had fewer ejaculations and longer interintromission intervals. Flupenthixol also antagonized the facilitative effects of apomorphine injections into the medial preoptic area. Flupenthixol and apomorphine produced only minor alterations in noncopulatory behaviors. The results suggest that dopamine receptors within the medial preoptic area are important in the regulation of masculine sexual behavior in the rat.