Behavioral and anatomical correlates of immunologically induced rejection of nigral xenografts.

Cell suspensions derived from the ventral mesencephalon of CD-1 mice were unilaterally transplanted into the striatum of neonatal Sprague-Dawley rats that had been bilaterally dopamine depleted. Thirty-eight percent of the grafts survived. Tyrosine-hydroxylase-immunoreactive neurons within the transplant innervated the host striatum with a dense fiber plexus. The grafts appeared to exert some degree of functional control over motor behavior in that these animals made contralateral rotations in response to amphetamine and tail pinch. In order to provide additional evidence that the motor behavior is associated with the transplant itself, the graft was removed. This was achieved by using a mouse skin graft to provoke an immunological response against the transplanted neural tissue. The immunological response resulted in the specific loss of the transplant with little or no damage to the surrounding neural tissue. The amount of rotation observed after tail pinch and amphetamine injection was severely affected by neural graft rejection. The loss of turning was associated most directly with the loss of tyrosine hydroxylase immunoreactivity within the transplant rather than with the massive reduction of tyrosine-hydroxylase-positive fibers in the ipsilateral host striatum. These data suggest that dopamine cells in mouse nigral grafts play an essential role in eliciting rotational behavior in neonatally dopamine depleted rats. They also show the value of skin grafting as a technique for specifically removing neural xenografts.

The development of striatal patch/matrix organization after prenatal methylazoxymethanol: a combined immunocytochemical and bromo-deoxy-uridine birthdating study.

The antimitotic drug methylazoxymethanol was used to destroy striatal patch neurons during their three-day-period of neurogenesis in the rat. Single or multiple injections of methylazoxymethanol were given during embryonic days 13-15, the period when patch neurons are known to undergo their final cell division. Methylazoxymethanol treatments produced a dramatic reduction in striatal volume. Immunocytochemical analysis revealed the continued presence of patches of neurons that were substance P-immunoreactive and devoid of calbindin and enkephalin immunoreactivity. Both the number of patches and relative volume occupied by patches was reduced in methylazoxymethanol-treated striata. Patch neurons could also be labelled by an intrastriatal injection of FluoroGold during the first postnatal week. The early ingrowth of nigrostriatal dopamine afferents was less noticeably patchy in the methylazoxymethanol-treated animals, in part owing to an overall increase in density. Large reductions in the number of neurons immunoreactive for choline acetyltransferase were observed, whereas NADPH diaphorase-stained neurons were not reduced unless methylazoxymethanol was given on embryonic day 15. Injections of bromo-deoxy-uridine, either during or after the 24 h that each methylazoxymethanol injection was considered to be effective, revealed that (i) some patch neurons continued to be generated in the 24-h period following methylazoxymethanol administration, and (ii) many patch neurons were generated after the effects of methylazoxymethanol had worn off. These findings demonstrate that it was impossible to completely eliminate the patches using methylazoxymethanol injections during the period of patch neurogenesis. However, methylazoxymethanol treatment during this time did produce a dramatic loss of cells and a relatively greater reduction in patch volume. Despite this disruption, the appropriate compartmentalization of neuroactive substances appeared to be maintained.

Development of dopamine innervation and turning behavior in dopamine-depleted infant rats receiving unilateral nigral transplants.

Three-day-old rats were bilaterally dopamine-depleted with 6-hydroxydopamine and 3 days later cell suspensions derived from the dopamine-rich ventral mesencephalic area were injected into the right rostral striatum. The transplants rapidly developed a substantial innervation of one striatum, so that by 15 days after transplantation (21 days of age) animals rotated away from the reinnervated side in response to amphetamine. The amount of turning correlated with the extent of innervation of the striatum as determined by tyrosine hydroxylase immunocytochemistry. By 25 days post-transplantation (31 days of age), animals turned in response to stress as well as amphetamine, although this later-developing phenomenon was not associated with any significant change in the extent of dopamine innervation. A second group of animals was bilaterally dopamine-depleted at 3 days of age, but transplantation with nigral cell suspensions was delayed until maturity. Partial reinnervation of the rostral striatum occurred with this delayed transplant paradigm, and turning to both amphetamine and stress commenced at 15 days post-transplantation. In contrast to animals receiving transplants shortly after lesioning, these animals began to turn spontaneously at about 20 days post-transplantation. The serotonin hyperinnervation that occurs following dopamine depletion in infancy was not altered by dopamine transplants made at either time. Results from both groups of transplant animals suggest that dopamine-rich transplants can provide substantial innervation that exerts some functional control over the striatum. This occurs despite the fact that neonatally dopamine-depleted rats, unlike adult-lesioned animals, survive quite well in the absence of striatal dopamine. However, the degree of incorporation into existing circuitry, and the types of regulation that result, may vary considerably depending on the age at which the tissue is transplanted.

Amphetamine sensitization of stress-induced turning in animals given unilateral dopamine transplants in infancy.

Infant rats given bilateral dopamine-depleting brain lesions and unilateral transplants of embryonic nigral tissue develop turning in response to both amphetamine and stress. However, stress-induced turning did not develop unless animals were previously exposed to amphetamine, and was greatest in animals exposed early to the drug. These findings suggest that amphetamine alters certain properties of the transplanted cells so as to enhance their functional capacity.

Amphetamine- and stress-induced turning after nigral transplants in neonatally dopamine-depleted rats.

Neonatally dopamine-depleted rats fail to show the deficits seen in similarly lesioned adults, and no longer appear to require dopamine. Nevertheless, unilateral nigral transplants provided extensive reinnervation and were capable of modifying motor patterns. Both amphetamine and external stressors elicited contraversive turning. This raises the question of whether extrinsic dopamine innervation supplements or overrides alternative compensatory mechanisms in the neonate.

Effects of prenatal cocaine exposure on the mesocorticolimbic dopamine system: an in vivo microdialysis study in the rat.

Microdialysis studies were conducted on prenatally saline-treated and prenatally cocaine-treated rats, either as pups (10-30 days old) or young adults (40-190 days old), to study the effects of prenatal cocaine exposure on the mesolimbic dopamine (DA) system. In the n. accumbens of saline-treated rats, basal dialysate concentrations of DA were similar in pups and adults; however, the levels of DA metabolites, DOPAC, HVA, and the serotonin metabolite, 5-HIAA, were markedly lower in pups. In pups, prenatal cocaine exposure led to basal dialysate levels of DA in the n. accumbens that were twice control levels; however, there was no difference in response to a period of intermittent tail pinch or an acute injection of cocaine (20 mg/kg). In the adult, basal levels of DA, DOPAC, HVA and 5-HIAA in n. accumbens did not differ across prenatal treatments. However, in prenatally cocaine-treated adults a cocaine injection led to an enhanced rise in extracellular DA compared to controls. In frontal cortex of adult rats, basal levels of DA, DOPAC and HVA did not differ across prenatal treatments; however, basal levels of 5-HIAA in this region were significantly elevated in prenatal-cocaine rats. No group differences were observed in the frontal cortex in response to either tail pinch or cocaine. Thus prenatal cocaine exposure produces an increase in basal extracellular DA in the n. accumbens of pups which returns to normal with aging. While this initial difference normalizes, prenatal cocaine exposure induces other persistent changes in adulthood.

Prenatal cocaine increases striatal serotonin innervation without altering the patch/matrix organization of intrinsic cell types.

The effect of prenatal cocaine on the anatomical development of the striatum was examined. The distribution and density of dopaminergic innervation of the striatum of animals exposed to cocaine during the second and third week of gestation was not noticeably different from prenatally saline-injected or untreated controls at any age. The patch/matrix organization of the striatum also appeared unaltered: neurons exhibiting dense substance P staining were localized to patches that overlapped dopamine terminal patches early in development, and enkephalin- and calbindin-immunoreactive neurons were found segregated to the matrix. Histochemical staining for acetylcholinesterase and NADPH diaphorase also revealed no differences between prenatally cocaine-treated and control brains. Whereas prenatal cocaine treatment failed to modify the basic compartmental organization of the striatum, it did lead to a hyperinnervation of serotonin-immunoreactive fibers which developed slowly after birth. Thus prenatal exposure to cocaine is capable of altering the ingrowth of serotonergic projections to the striatum while producing no change in the organization of neurons intrinsic to the striatum.

Fluoro-Gold reveals patches of striatal neurons during development.

Patches of densely-labelled striatal neurons were observed following intrastriatal injection of the fluorescent dye Fluoro-Gold into rats younger than 10 days of age. These cell clusters showed precise overlap with islands of dense dopamine innervation seen only during the first week of life. The patches of labelled cells were still visible in adulthood, and were not abolished by 6-hydroxydopamine treatment before or after the Fluoro-Gold injection. Neonatal Fluoro-Gold injection may be a unique way of marking the patch compartment of the striatum that remains visible in adulthood.

Prenatal cocaine alters later sensitivity to cocaine-induced seizures.

Rats that had been prenatally exposed to cocaine were tested later in life for their sensitivity to cocaine-kindled seizures and acute cocaine-induced seizures. When treated daily with cocaine, beginning at one month of age, males prenatally exposed to 40 mg/kg cocaine developed seizures in a fewer number of days than those prenatally exposed to saline. Prenatally cocaine-treated females did not seize more rapidly than controls in the cocaine kindling paradigm; however, they were more susceptible to seizures in response to an acute high dose of cocaine. These results suggest that rats prenatally cocaine-treated are more sensitive to the seizure-producing effects of cocaine later in life, and this enhanced sensitivity is differentially expressed in males and females.

Audiogenic seizures induce c-fos in a model of developmental epilepsy.

In rats made susceptible to audiogenic seizures by exposing them to an intense noise at a critical time during development, subsequent noise exposure elicited seizures and induced the proto-oncogene c-fos in auditory regions of the brain. Cells showing Fos-like immunoreactivity were especially dense in dorsal and external cortices of the inferior colliculus, and were nearly absent after pretreatment with the N-methyl-D-aspartate (NMDA) antagonist MK-801. Noise exposure alone (i.e. no seizure) produced a localized zone of c-fos induction within the inferior colliculus, but only when presented during the time period when susceptibility to audiogenic seizures can be most effectively induced.

Development of striatal compartmentalization following pre- or postnatal dopamine depletion.

Nigrostriatal dopamine (DA) projections terminate in distinct patches during the late prenatal and early postnatal period in the rat. During the first postnatal week, patches of DA fibers overlap with clusters of striatal neurons that share several identified characteristics. The early segregation of striatal cell types into either these patches or the surrounding matrix becomes a permanent organizational feature of the striatum. In order to determine whether the heterogeneous distribution of DA influences the formation of cellular patches, the developmental organization of chemically identifiable cell types was examined in normal rats and in rats DA depleted as infants (0 or 3 d) or in utero (embryonic days 17-18). During the first postnatal week, corresponding patches of DA afferents and substance P (SP)-immunoreactive neurons existed in the striatum of normal animals, and AChE-positive zones overlapped these patches in the lateral striatum. Injection of 6-hydroxydopamine into the lateral ventricles of fetal or infant rats produced a dramatic loss of striatal DA terminals. Neither the patchy distribution of SP-immunoreactive neurons nor the distinctive pattern of AChE staining present during the first 2 postnatal weeks was disrupted. During the third postnatal week, cells immunoreactive for leu-enkephalin or calbindin-D28k were confined to the matrix compartment, and this compartmentalization was also not noticeably changed by pre- or postnatal DA depletion. In adult animals, overlapping patches of leu-enkephalin- and SP-immunoreactive fibers were observed, regardless of whether any DA terminals remained. Thus, the basic organization of the striatal patch and matrix compartments develops normally in the absence of DA innervation through much of the formative period. Although these observations do not completely dismiss the possibility that the first DA afferents to appear in the striatal primordia influence contracted striatal cells to develop the patch phenotype, they suggest that the patchy distribution of DA afferents may be secondary to the early clustering of striatal neurons forming the patch compartment.

Striatal c-fos induction by drugs and stress in neonatally dopamine-depleted rats given nigral transplants: importance of NMDA activation and relevance to sensitization phenomena.

Induction of the proto-oncogene c-fos occurred in cells of the striatum in response to stimuli that are known to release dopamine (DA). As revealed by fos immunocytochemistry, amphetamine (AMPH) produced c-fos induction in many cells of the medial two-thirds of the striatum of normal rats, with patchy labeling in the lateral third. The lateral patches were found to be coincident with patches of striatal neurons lacking calbindin immunoreactivity. In animals DA-depleted at birth, few fos-immunoreactive neurons were present in response to AMPH. In those with unilateral transplants of DA-rich mesencephalic tissue, c-fos induction was greater on the transplanted side. The DA D1 antagonist SCH 23390 completely blocked c-fos induction in all animals. The N-methyl-D-aspartate antagonist MK-801 also blocked c-fos induction by AMPH within the medial striatum, but intensified c-fos induction laterally in those animals with DA innervation. A second set of experiments examined the functional importance of c-fos induction in the AMPH sensitization of turning behavior that occurs in these animals. Both AMPH and stress produced turning, but only AMPH produced widespread c-fos induction, and stress-induced turning only occurred after exposure to AMPH. Treatment with MK-801 prior to AMPH administration blocked the subsequent development of stress-induced turning. Whereas a high dose of MK-801 (1.0 mg/kg) completely blocked c-fos induction, a lower dose (0.1 mg/kg) blocked c-fos induction in controls, but left patches of fos-immunoreactive neurons in lesioned animals given transplants. Thus the sensitization of transplant-related behaviors is NMDA dependent and associated with c-fos induction in host striatal neurons.

Enhanced efficacy of nigral-striatal cotransplants in bilaterally dopamine-depleted rats: an anatomical and behavioral analysis.

When embryonic ventral mesencephalic tissue containing nigral dopamine (DA) neurons is transplanted into adult DA-depleted striatum, synaptic connections form and behavioral effects are observed. This study investigated the cotransplantation of embryonic striatal tissue as a means of enhancing the innervation, survival, and functional effects of nigral transplants. Rats neonatally DA-depleted, via bilateral intraventricular injections of 6-hydroxydopamine, developed turning in response to amphetamine and stress following unilateral transplantation of either nigral or combination nigral-striatal cell suspensions. Animals with cotransplants developed higher levels of turning to both stimuli and maintained these responses for a longer period of time post-transplantation, when compared with animals receiving transplants of nigral cells alone. In addition, these combination transplants required fewer dopaminergic cells to produce a strong behavioral effect on the host. Dense patches of tyrosine hydroxylase (TH)-immunoreactive fibers were observed within the cotransplants, yet no greater outgrowth of DA fibers into host striatum was detected. Amphetamine produced widespread induction of the immediate-early gene c-fos in cells of host striatum that extended beyond the transplant-derived DA innervation. After both amphetamine and stress, Fos protein was found within both types of transplants, but these Fos-immunoreactive cells did not colocalize with TH-immunoreactive cells nor dense TH-immunoreactive patches within the grafts. Thus, cotransplanted embryonic striatal tissue augments the effects of ventral mesencephalic transplants, possibly by providing a trophic influence that enhances the function of the DA cells without increasing cell survival.