Postnatal development of caudate input neurons in the cat.

Lectin-bound horseradish peroxidase (WG-HRP) was pressure-injected into the caudate nucleus (Cd) of neonatal (less than 24 hours of age) and adult cats in order to assess the postnatal development of monosynaptic Cd input neurons. Tissue was processed for peroxidase activity with a benzidine dihydrochloride chromagen. The injection of WG-HRP produced relatively similar labelled zones of marker uptake in the caudate nuclei of both neonates and adults. Similar axonal projections were also labelled in both age groups. While many characteristics of retrogradely labelled CD input neurons were apparently constant throughout postnatal life, each of these features had a particular developmental modification. (1) Regardless of age, neuronal somata that projected to the CD were located in the neocortex, thalamus, substantia nigra, mesencephalic raphe nuclei, and globus pallidus. In each of these brain sites, labelled CD input neurons appeared to migrate postnatally. (2) The Cd afferent axons originated from the same neuronal lines in neonates and adults--small-to-medium-sized cortical neurons and medium-sized-to-large fusiform cells in all other brain sites. In each of the brain sites, labelled neurons displayed marked postnatal somatic growth. (3) In both age groups, there was a characteristic intrasomatic reaction product density in the labelled neurons located in each brain site (substantia nigra greater than thalamus = raphe = globus pallidus greater than cortex). In each of these brain sites, the intrasomatic reaction product density was less in neonates than in adults.

Phenylcyclohexylamine: effect of a metabolite of phencyclidine on the efflux of dopamine in the rat.

The effect of phenylcyclohexylamine (PCA) on the efflux of dopamine (DA) in the neostriatum was examined using in vivo electrochemical techniques. Phenylcyclohexylamine produced a long-lasting dose-dependent biphasic effect on the efflux of DA in the rat. This response, to one of the major metabolites of phencyclidine, was similar in duration to but less potent than that seen with phencyclidine.

Effects of phencyclidine on extracellular levels of dopamine, dihydroxyphenylacetic acid and homovanillic acid in conscious and anesthetized rats.

Dose-dependent effects of phencyclidine on extracellular levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the neostriatum were studied in both urethane-anesthetized and conscious rats. In vivo microdialysis was used to collect 10 min samples that were analyzed for levels of DA, DOPAC and HVA, using high-performance liquid chromatography with electrochemical detection (HPLC-EC). In both the anesthetized and conscious preparations, 20 mg/kg of phencyclidine produced an increase in extracellular levels of DA, 10 mg/kg resulted in no change, while 1 mg/kg produced a slow decrease. In the anesthetized animals phencyclidine did not have a significant effect on levels of DOPAC or HVA, but in the conscious animals phencyclidine produced a dose-dependent decrease in levels of DOPAC and HVA. The increase in levels of DA could be the result of increased release of DA or inhibition of the uptake of DA. The decrease in levels of DOPAC and HVA, at the 1 mg/kg dose, could result from a decrease in the synthesis of DA that is offset at the 10 and 20 mg/kg doses by opposing mechanisms.

Declines in stimulated striatal dopamine release over the first 32 h following microdialysis probe insertion: generalization across releasing mechanisms.

In a recent paper [R.R. Holson, J.F. Bowyer, P. Clausing, B. Gough, Methamphetamine-stimulated striatal dopamine release declines rapidly over time following microdialysis probe insertion, Brain Res. 739 (1996) 301-307] we reported that methamphetamine-stimulated striatal dopamine release declined rapidly over the first eight hours following microdialysis probe insertion. This decline was strictly a function of time post-probe implantation, and not due to tolerance or desensitization. To further examine this phenomenon, we subjected rats to three brief pulses of several DA-releasing compounds at 2, 4 and 6 h post-probe insertion, and compared these results to those caused by a single pulse 6 h post-insertion, or in some cases to pulses given more than 24 h post-insertion. We found that when buproprion, a dopamine reuptake blocker, was infused briefly into the striatum via the microdialysis probe, there was a pronounced drop in the amount of dopamine released at 6 h vs. 2 h post-insertion; this drop was not due to repeated exposure, since dopamine release at 6 h post-insertion was the same for a single pulse, or when preceded by two earlier pulses. Twenty-four hours later, buproprion-stimulated dopamine release was still lower, but did not appear to drop further thereafter. Potassium-stimulated dopamine release, on the other hand, dropped rapidly over the first 8 h post-insertion, and this decline continued throughout the 24-32 h interval post-insertion. Similarly, a single i.p. injection of 0.5 mg/kg haloperidol released three times as much dopamine when given two compared to six hours post-implantation. Both bupropion- and potassium-stimulated dopamine release were accompanied by declines in extracellular DOPAC concentrations, and these declines were the same 2 or 26 h post-insertion. In contrast, haloperidol exposure increased extracellular DOPAC, and this haloperidol-stimulated DOPAC increase was also greatly attenuated at 6 compared to 2 h post-insertion. We conclude that there is a general decline over time post-probe implantation in the ability of the striatal dopamine system to release dopamine, and perhaps to increase dopamine synthesis, in response to pharmacological challenges.

The ontogeny of amphetamine-induced dopamine release in the caudate-putamen of the rat.

Amphetamine-induced dopamine (DA) release in the caudate-putamen of adult rats was compared with that in the 35-36-day-old and 21-22-day-old rat pup, using in vivo voltammetry. In the adult and 35-36-day groups, 1.0 mg/kg amphetamine (AMP) produced a significant increase in DA release, while 0.1 mg/kg produced no significant change in DA release. In the 21-22-day group, 1.0 mg/kg AMP produced a slight increase, followed immediately by a significant decrease in DA release. Similarly, at a dose of 0.1 mg/kg, AMP produced a significant decrease in DA release. This decrease was greater than that seen after the 1.0 mg/kg dose of AMP. Tyramine produced no significant change in DA release, however, it served as a control for peripheral cardiovascular effects. These data suggest that AMP-induced DA release in the caudate-putamen is mature by postnatal day 35. The AMP-induced decrease in DA release found in the 21-22-day group is not due to either the cardiovascular effects of AMP or to a depletion of DA content. This decrease in neostriatal DA release may be due to a decrease in the neuronal firing of nigrostriatal DA neurons that is caused by an AMP-induced increase in dendritic DA release in the substantia nigra, exerting an inhibitory effect through DA autoreceptors.

Physiological and morphological analyses of ventral anterior and ventral lateral thalamic neurons in the cat.

Intracellular recordings were made from ventral anterior and ventral lateral (VA-VL) thalamic neurons in the cat. VA-VL neurons were tested for responsiveness to activation of cortical, pallidal and cerebellar afferents, and were identified morphologically by intracellular injection of HRP. Orthodromic activation of cortical and pallidal afferents produced primarily an initial inhibition (due in part to oligosynaptic circuitry) while activation of cerebellar afferents produced an initial excitation in the majority of neurons tested. Antidromic activation of thalamocortical relay neurons was observed in 32% of the neurons tested. Neurons showing short latency responses to activation of globus pallidus-entopeduncular nucleus and cerebellar peduncle were concentrated in the medical and ventral portions of the VA-VL complex, respectively. Neurons showing short latency responses to activation of the neocortex were located throughout the entire extent of the VA-VL complex. Only 3% of the neurons tested showed short-latency convergence of cortical, pallidal and cerebellar afferents. In contrast, 53% of neurons tested showed long latency triple convergence. Eight VA-VL neurons were stained intracellularly with HRP. Based on dendritic morphology, the labeled neurons were separated into two types: a stellate type with dendrites that spread radially from the soma, and a fusiform type with dendrites that were oriented mainly parallel to the long axis of the soma. Both types of neuron were aspiny although the dendrites of the stellate cells exhibited short appendages.

Inhibition of dopamine release by methylenedioxymethamphetamine is mediated by serotonin.

(+/-)-3,4-Methylenedioxymethamphetamine (MDMA), at doses of 0.1, 1 and 10 mg/kg, produced a long-lasting decrease in extracellular dopamine concentration in the neostriatum of anesthetized rats, as measured by in vivo voltammetry. Since MDMA has been shown to release serotonin from rat brain slices and synaptosomes, we examined the possibility that increased serotonin release might be the cause of the decrease in dopamine release. Rats were treated with d,l-p-chloroamphetamine seven days prior to acute MDMA administration. Rats pretreated with p-chloroamphetamine, which produced a marked decrease in serotonin content, showed no significant decrease in extracellular dopamine concentration when administered 10 mg/kg MDMA. These data suggest that MDMA produces a significant decrease in dopamine release when administered acutely, and that this decrease is an indirect effect mediated by an increase in serotonin release.

The development of D2 autoreceptor-mediated modulation of K(+)-evoked dopamine release in the neostriatum.

A within-subject dose-response analysis was conducted by locally perfusing increasing concentrations (0.1, 1, 10 and 100 microM) of the selective D2 agonist quinpirole via a microdialysis probe into the neostriatum of urethane-anesthetized rat pups 5, 10-11, 15-16 and 21-22 days of age and adult rats. In Expt. 1, K(+)-evoked dopamine release was significantly decreased by quinpirole relative to the vehicle control group for each age in a dose-dependent manner. The maximum effect of quinpirole was not influenced by acute tolerance or the length of the experiment (Expt. 2). Finally, the effect of quinpirole (10 microM) was blocked by the addition of the selective D2 antagonist (-)-sulpiride (100 microM) to the perfusion solution (Expt. 3). These results support and extend previous research that suggests that presynaptic D2 autoreceptors in the neostriatum are able to modulate K(+)-evoked dopamine release in vivo by postnatal day 5 in the rat.

The effects of bupropion in vivo in the neostriatum of 5-day-old and adult rats.

Infusion of six concentrations of the dopamine uptake inhibitor bupropion into the neostriatum increased extracellular dopamine in a dose-dependent manner in 5-day-old and adult rats. There was no age-related difference when calculated as a percentage of predrug dopamine baseline levels, but the absolute increase of dopamine was greater in the adult rats. Bupropion had only a minor effect on extracellular levels of DOPAC.

Effect of milk on dopamine release in the newborn rat: an in vivo microdialysis study.

Newborn rats exhibit a rich behavioral repertoire to access the nipple and obtain milk. In older pups, catecholamines including dopamine (DA) mediate the behavioral effects of milk. In the present study, pups were delivered at term by caesarean section and instrumented with the microdialysis probe. Microdialysis samples were collected at 15 min intervals and K(+)-evoked levels of DA were measured with HPLC-ED. Pups received either no infusion, single or multiple intraoral infusions of saline or milk during subsequent samples. A decrease in K(+)-evoked DA release was evident after the first infusion in all subjects. Repeated milk infusions continued to reduce levels of extracellular DA, which remained evident 30 min after the last milk infusion. The rat neonate's first exposure to milk exerts lasting effects on neostriatal DA activity in the absence of prior suckling experience.

3H-xylamine binds to presynaptic rat striatal membranes.

3H-xylamine (3H-XYL), an irreversible catecholamine uptake inhibitor, was incubated with rat striatal synaptosomes, and the membrane fraction was examined by fluorography of a sodium dodecyl sulfate-polyacrylamide gel. A number of peptides were labeled. To determine their location, the striatal dopaminergic presynaptic nerve terminals were destroyed by unilateral electrolytic lesions through the nigrostriatal fibers prior to 3H-XYL exposure. The 3H-XYL bound to membranes from lesioned striata was about 29% of that bound to control membranes, which is consistent with the 83% reduction in dopamine (DA) uptake and the 68% reduction in DA content in the lesioned tissue. The decrease in peptide-bound 3H-XYL paralleled the decrease in DA content, with the exception of a 45% decrease in binding to a 45K peptide. These data show that 3H-XYL binding is predominantly localized in the dopaminergic presynaptic nerve terminals of the striatum.

Behavioral effects of low-dose gestational day 11-13 retinoic acid exposure.

In a comparison article we report that maternal PO exposure to 2.5 mg/kg all-trans retinoic acid (RA) daily for 3 consecutive days over gestational days (GD) 11-13 produces a 10% reduction in weight of cerebellum at 4 weeks of age, not accompanied by other malformations. Here we report the results of a preliminary behavioral analysis of offspring exposed gestationally to RA as above. Exposed dams were allowed to deliver normally, and litters were culled to eight pups (4 +/- 1 of each sex) at birth. Both male and female offspring were tested prior to weaning on GD 21. Thereafter females were killed on postnatal day (PND) 28 for verification of RA effects on regional brain weight, and all subsequent behavioral testing was conducted on males. Preweaning tests were restricted to negative geotaxis (PND 8-9) and open field activity (PND 22). Postweaning tests included open field activity (PND 43), auditory startle response (three times, on PNDs 22, 43, and 84), 2-week activity in residential running wheels (PNDs 62-76), complex maze performance for 5 consecutive days (PND 83-87), emergence latency (PND 106), and assessment of the behavioral response to an amphetamine challenge (PND 107). Males were then killed on PND 108 for verification of RA effects on regional brain weights. In this study, RA reduced weight of cerebellum but not striatum. Cerebellar weight was 92% of control values in PND 28 females, and this weight difference had diminished to 95% of control weight by PND 108 in males. There were no treatment effects on negative geotaxis, activity in a small open field, auditory startle amplitude, or latency to enter an illuminated alley from a dark chamber. Maze learning occurred at levels equal to or slightly better than controls. Running wheel activity was enhanced by RA exposure, whereas activity in response to an amphetamine challenge was reduced by such exposure. We conclude that RA doses low enough to produce mild weight reductions in cerebellum, without attendant malformations, can alter behavior. The precise nature of these alterations remains to be elucidated, but the findings reported here suggest that effects may be more pronounced on activity than on learning.

A behavioral and neuroanatomical investigation of the lethality caused by gestational day 11-13 retinoic acid exposure.

In a companion article, we report that there is a sensitive period for all-trans retinoic acid (RA) lethality on gestational days (GD) 11-13. When dams were given 10 mg/kg RA daily for 3 consecutive days on GD 11-13, a number of pups were found dead in the home cage on the day of birth, and the remainder inevitably died due to an apparent inability to nurse. Here we report a set of experiments further investigating these effects. Dams were exposed PO to 10 mg/kg RA or oil vehicle on GD 11-13. Fetuses were removed by Cesarean section on the afternoon of GD 21, culled, and fostered to non-treated dams that had given normal vaginal delivery a day earlier. Maternal behavior was observed for the first 6 h after fostering. The next morning all surviving pups were given a brief behavioral evaluation, including the ability to attach to the nipple of anesthetized foster dams. At the time of C-section, culls were killed and brains were quickly removed and placed in fixative. A series of paraffin-embedded, cresyl-violet-stained serial sections of a representative brain stem from each litter was prepared. RA exposure did not increase fetal mortality. Treated litters were as large as controls, and virtually all treated fetuses were alive in utero. However, unlike controls, some treated fetuses appeared to have difficulty in initiating spontaneous breathing when delivered by C-section, and considerable physical stimulation was required before normal breathing began. As in the previous report, RA-exposed pups did not have milk in their stomachs after 18 h on the foster dam; further, they did not attach to the maternal nipple, and they had greater difficulty than controls in maintaining an upright posture. Examination of serial sections of the medulla indicated that the hypoglossal nucleus appeared grossly normal in the RA-exposed pups. In contrast, the inferior olive and the area postrema were affected by RA exposure. Both nuclei were normally located, but exhibited reduced cell density and/or intensity of staining. In the inferior olive the dorsal and principal nuclei were primarily affected, to the degree that about one quarter of treated brains had no identifiable principal nucleus. We conclude that RA exposure on GD 11-13 causes abnormal development of cell-dense regions of the medial medulla, and these abnormalities may account for the difficulty these animals experience in beginning spontaneous breathing and in nursing. These breathing and nursing problems in turn almost certainly account for the high mortality seen during natural birth and for the subsequent failure to thrive, respectively.

Minimal behavioral effects from moderate postnatal lead treatment in rats.

Developmental lead exposure continues to be a worldwide problem. This study investigated the behavioral effects resulting from developmental lead treatment in rats with corresponding physiological measures of lead exposure. Sprague-Dawley rats were treated with 350 ppm lead acetate from birth to weaning via the dam's drinking water. Behavioral measures assessed in the offspring included residential activity tests, complex maze performance, acoustic startle response, emergence behavior (light/dark preference), prepulse inhibition, and ethological assessments of play, dominance, and burrowing. Pb blood levels averaged 53 microg/dl in the dam at the time of offspring weaning and 46 microg/dl in weanling female offspring. Pb levels averaged 277 ng/g and 32 microg/g in the brain and bone, respectively, of female offspring at weaning. No behavioral assessment indicated any lead-related functional alterations nor were there any statistically significant differences when the lead-treated group was restricted to rats in those litters that were above the median Pb blood lead level at weaning. These results indicate that any lead-related functional alterations at this dose may be subtle and require a sufficient demand on the system for detection.

Gestational retinoic acid exposure: a sensitive period for effects on neonatal mortality and cerebellar development.

This is the first in a series of studies investigating the developmental stage-specific neurobehavioral effects of all-trans retinoic acid (RA) exposure. Because high doses of this compound are known to be lethal to the developing organism, we first conducted a dose-response study to identify RA doses that produce low enough levels of gestational/postnatal mortality to make a behavioral analysis possible in survivors. Secondarily, at doses found to produce sufficient survivors on PND 28, effects on body and regional brain weights were examined. Finally, at these doses, effects on somatic malformations were evaluated. Four separate exposure periods were analyzed: gestational days (GD) 8 through 10, 11 through 13, 14 through 16, or postnatal days (PND) 3 through 5. In the postnatal exposure period rat pups were injected (s.c.) with three consecutive daily doses of 0, 5, 10, or 20 mg/kg RA on PND 3 through 5. This postnatal exposure had no detectable effect on survival, body or brain weight. In contrast, there was a marked sensitivity to RA in the GD 11-13 group. Many pups from dams given 10 mg/kg RA PO on GD 11-13 were found dead in the cage on the day of birth, and all surviving pups died within 4 days of birth. Examination of milkbands revealed no evidence of effective suckling in these short-term survivors. The same 10 mg/kg dose at GD 8-10 or GD 14-16 produced much lower mortality and pups appeared to suckle normally. To produce adequate PND 28 survival in the GD 11-13 group, it was necessary to reduce dosage to 2.5 mg/kg daily. Even this lower exposure produced effects on PND 28 body and brain weight, significantly lowering weights of body (84% of control), whole brain (94%), and cerebellum (90%). Cerebellar weight was also depressed as percent of whole brain weight, suggesting an effect focused specifically on this region. RA at 10 or 12.5 mg/kg over GD 14-16 also reduced cerebellar weight (92% and 91% of control, respectively). Thus, exposure on GD 14-16 had effects similar to those seen at GD 11-13, but only at considerably higher doses. In contrast, exposure to RA on GD 8-10 did not affect whole body or brain weight, and of eight brain regions examined, only brain stem weight was reduced (91% of control). The GD 8-10 exposure also differed substantially from later exposures in that it was the only treatment to produce substantial malformations, including exencephaly, eye and skeletal defects. We conclude that gestational exposure to RA produces lethality and regional brain stunting that is dose and developmental stage specific, with a pronounced sensitive period on GD 11-13. In contrast, the GD 8-10 period is most sensitive for production of malformations, albeit at somewhat higher doses.

Calcium dependency and tetrodotoxin sensitivity of neostriatal dopamine release in 5-day-old and adult rats as measured by in vivo microdialysis.

The calcium dependency and tetrodotoxin sensitivity of extracellular dopamine levels were assessed by microdialysis in the neostriatum of 5-day-old rat pups and were compared with those obtained in adult rats. The removal of calcium from the dialysate reduced spontaneous levels of extracellular dopamine to 20% of normal in the 5-day-old pups and to 10% of normal in the adults. Calcium-free dialysate also decreased potassium-evoked dopamine release to approximately 20% of baseline in both ages. Furthermore, the addition of tetrodotoxin to the dialysate decreased spontaneous levels of extracellular dopamine to 10% of baseline in both ages. The effects of calcium removal and the addition of tetrodotoxin on extracellular levels of the dopamine metabolite 3,4-dihydroxyphenylacetic acid were less pronounced. The results of this study demonstrate that extracellular levels of dopamine sampled by microdialysis in rats as young as 5 days of age are both calcium dependent and tetrodotoxin sensitive; thus, they are derived from neuronal activity and not from injury caused by acute implantation of the probe. Other age-related differences support the hypothesis that dopamine release and turnover is greater in immature rats and may represent a form of compensation for incomplete dopamine nerve terminal ingrowth.

Early postnatal x-irradiation of the hippocampus and discrimination learning in adult rats.

Rats with X-irradiation-produced degranulation of the hippocampal dentate gyrus were trained in the acquisition and reversal of simultaneous visual and tactile discriminations in a T-maze. These experiments employed the same treatment, apparatus, and procedure but varied in task difficulty. In the brightness and roughness discriminations, the irradiated rats were not handicapped in acquiring or reversing discriminations of low or low-moderate task difficulty. However, these rats were handicapped in acquiring and reversing discriminations of moderate and high task difficulty. In a Black/White discrimination, in which the stimuli were restricted to the goal-arm walls, the irradiated rats were handicapped in the acquisition (low task difficulty) and reversal (moderate task difficulty) phases of the task. These results suggest that the irradiated rats were not handicapped when the noticeability of the stimuli was high, irrespective of modality used, but were handicapped when the noticeability of the stimuli was low. In addition, these results are consistent with the hypothesis that rats with hippocampal damage are inattentive due to hyperactivity.

The ontogeny of apomorphine-induced alterations of neostriatal dopamine release: effects on potassium-evoked release.

The effects of apomorphine (0.05, 0.1, and 1.0 mg/kg, s.c.) on K(+)-evoked dopamine release were studied through the use of in vivo microdialysis in the neostriatum of developing and adult rats. Fifteen-minute samples were collected from urethane-anesthetized rats 5, 10-11, 21-22, 35-36 days of age, and adults, and quantified by high performance liquid chromatography with electrochemical detection. Apomorphine attenuated K(+)-evoked dopamine release in all age groups, suggesting that the dopamine autoreceptor modulating release in the neostriatum is functional by 5 days of age. A dose-response effect of apomorphine was observed in all age groups except at 5 and 10 days of age. Absolute levels of extracellular dopamine were significantly lower at 5 and 10 days of age compared with the other ages, and the effectiveness of a high-K+ artificial cerebrospinal fluid to evoke dopamine release increased with age.

Effects of (-)-sulpiride on dopamine release in striatum of developing rats: degree of depolarization influences responsiveness.

The purpose of this study was to determine the effects of localized delivery of the D2 antagonist (-)-sulpiride (via microdialysis) on spontaneous and evoked dopamine release in the neostriatum of urethane-anesthetized rats 5, 10, 15, 21, and 70 days of age. Sulpiride increased spontaneous dopamine release approximately threefold relative to baseline measures, and this effect decreased with maturation. The relationship between sulpiride- and potassium-evoked release was complex; sulpiride increased evoked dopamine outflow at 5, 10, and 15 days of age. At 21 and 70 days of age, however, the effects of sulpiride were inversely related to the degree of stimulation with potassium. Furthermore, the D2 agonist quinpirole (100 microM) reversed the effects of sulpiride (10 microM), suggesting receptor mediation. These experiments demonstrate that the maturational decline in the efficacy and potency of D2 antagonism appears to be related to the degree of stimulation at the nerve terminal.