Abigail Snyder-Keller

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.

Prenatal cocaine exposure.

Abstract: Cocaine abuse is a significant problem not only in the general population but also among pregnant women. Since cocaine readily crosses the placenta and is metabolized slowly in fetuses, they can be exposed to significant levels of cocaine for long periods. In humans the most common consequences of cocaine abuse during pregnancy include premature birth, lower birth weight, respiratory distress, bowel infarctions, cerebral infarctions, reduced head circumference, and increased risk of seizures. Behaviorally these new‐borns show an increased degree of “tremulousness,” crying and irritability, and are over‐reactive to environmental stimuli. Within a month these behaviors have recovered dramatically, but not to normal levels. Thus while there are a number of abnormalities associated with cocaine‐exposed neonates, they are not imminently debilitating or life‐threatening. However, the long‐term consequences of this prenatal cocaine exposure remain to be elucidated. We have examined a rat model for neurochemical, neuroanatomical and behavioral changes resulting from prenatal cocaine exposure. Since cocaine is known to act by blocking the inactivation of the neurotransmitters dopamine, serotonin and norepinephrine, our studies have focused on brain dopamine (DA) and serotonin (5‐HT) pathways. In this model system we have found neurochemical changes that are present at birth and that return to normal as the rat ages‐similar to the recovery observed in infants. However, there are other neurochemical, anatomical and behavioral changes that persist after birth which may provide insights into the long‐term consequences. It is hoped that by understanding the changes occurring in this rat model we will be better prepared to devise pharmacological interventions to circumvent the secondary consequences of prenatal cocaine exposure. These consequences might include increased susceptibility to drug addiction, seizures, depression, schizophrenia, Parkinsons disease, etc.

Development of frequency-selective domains in inferior colliculus of normal and neonatally noise-exposed rats

Topographic patterns of pure-tone responses in inferior colliculus (IC) of Wistar rats were mapped using immunohistochemical staining for the nuclear protein Fos, the translation product of the c-fos proto-oncogene. Patterns were compared in ICs of immature and mature rats and in mature rats which experienced auditory deprivation beginning on day 14, an age near the developmental onset of hearing. Neonatal hearing losses, caused here by exposure to potentially deafening noise, are known to result in audiogenic seizure susceptibility in neonatal rats. These seizures can be triggered only by high-frequency stimuli and are believed to be initiated in IC. Thus, it seemed possible that susceptibility might depend on derangements of topographic frequency representation due to neonatal auditory deprivation. The band-like frequency-response domains, characteristic of adult IC, were found to be poorly differentiated in ICs of immature rats. On day 12, only lower-frequency stimuli induced discrete bands of Fos immunoreactivity while responses to higher frequencies remained exceptionally diffuse within ventral portions of IC. Only after day 24 did responses to the highest frequencies also appear mature. Furthermore, most significantly, adult rats which were transiently deafened on day 14, retained the more voluminous response patterns which were characteristic of immature IC. Because frequency selectivity in cochlea also develops by a low-to-high frequency sequence, results are consistent with a hypothesis that topographic organization arises in IC by an activity-dependent process. Whereas neonatal noise exposure also conferred audiogenic seizure susceptibility, it appears the arrest of tonotopic organization of IC is the probable basis of this reflex epilepsy.

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.

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.

Glutamate receptor subtypes localize to patches in the developing striatum

The distribution of glutamate receptors in the developing striatum of the rat was studied using antibodies specific to AMPA and NMDA subtypes. Immunocytochemistry revealed a greater density of GluR1, GluR2/3, NMDAR1, and NMDAR2A/2B receptors in patches that matched the patches of substance P-immunoreactive neurons and dopaminergic terminals. GluR1-immunoreactive patches were the most distinctive and were present already at embryonic day 19.

Compartmental expression of trkB receptor protein in the developing striatum.

To investigate the role of neurotrophins in the initial formation of striatal patch versus matrix, the spatial and temporal expression of trkB receptors was examined using immunohistochemistry. Polyclonal antibodies, against the C-terminus or the tyrosine kinase domain, revealed trkB-immunoreactive cells and fibers localized to patches beginning on embryonic day 19 in the rat, which co-localized with patchy dopamine fibers, substance P-immunoreactive neurons and glutamate receptors. Patchy striatal trkB expression was maintained after lesioning the nigrostriatal dopamine system. The patchy trkB distribution persisted through postnatal day 14, then became more homogeneous at the same time that nigrostriatal afferents become homogeneous. Later in development, trkB immunoreactivity was most intense in a subpopulation of large striatal cells that were similar in size and frequency to those immunoreactive for choline acetyltransferase. The spatiotemporal expression of trkB receptor in phenotypically distinct striatal patches, as well as evidence that neurotrophins regulate expression of neuronal phenotypic markers during development, may indicate a convergence of neurotrophins and afferent innervation on to future patch cells that may regulate the establishment of striatal compartmentalization.

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.

Co-transplantation of Fetal Lateral Ganglionic Eminence and Ventral Mesencephalon Can Augment Function and Development of Intrastriatal Transplants☆

Methods to increase the development and sustained function of embryonic mesencephalic dopamine cells after transplantation into dopamine (DA)-depleted striatum are currently under investigation. Elements that are crucial for the maturation and connectivity of neurons during normal development of the brain may also play a role in the development and integration of grafted embryonic tissue. Based on in vitro and in vivo observations of the enhancing effects of striatal tissue on nigral dopaminergic cell development and survival, we demonstrate that inclusion of embryonic striatal cells, specifically from the lateral ganglionic eminence (LGE), produces dopaminergic transplants with augmented functional effects. Rats neonatally DA-depleted and co-transplanted with embryonic nigral and LGE cells developed improved functional outcome when compared with animals receiving only nigral cells, and they required the transplantation of fewer nigral cells to produce a strong behavioral effect. Anatomically, the inclusion of LGE cells produced increased DA cell survival, a higher density of reinnervation into the DA-depleted host striatum, and patches of DA fibers within the co-transplants. There were also an increased number of host striatal cells which induced the immediate-early gene c-fos in co-transplanted animals compared to animals receiving nigral cells alone, indicating a higher degree of host-cell activation. The ability to enhance function, cell survival, reinnervation, and host activation with nigral-striatal co-transplants in the presence of fewer nigral cells supports the hypothesis of a trophic influence of striatal cells on nigral DA cells.

Development of striatal patch/matrix organization in organotypic co-cultures of perinatal striatum, cortex and substantia nigra

Organotypic cultures of fetal or early postnatal striatum were used to assess striatal patch formation and maintenance in the presence or absence of dopaminergic and glutamatergic influences. Vibratome-cut slices of the striatum prepared from embryonic day 19 to postnatal day 4 rat pups were maintained in static culture on clear membrane inserts in Dulbeccos modified Eagles medium/F12 (1:1) with 20% horse serum. Some were co-cultured with embryonic day 12-16 ventral mesencephalon and/or embryonic day 19 to postnatal day 4 cortex, which produced a dense dopaminergic innervation and a modest cortical innervation. Donors of striatal and cortical tissue were previously injected with bromo-deoxyuridine (BrdU) on embryonic days 13 and 14 in order to label striatal neurons destined to populate the patch compartment of the striatum. Patches of BrdU-immunoreactive cells were maintained in organotypic cultures of late prenatal (embryonic days 20-22) or early postnatal striatum in the absence of nigral dopaminergic or cortical glutamatergic influences. In slices taken from embryonic day 19 fetuses prior to the time of in vivo patch formation, patches were observed to form after 10 days in vitro, in 39% of nigral-striatal co-cultures compared to 6% of striatal slices cultured alone or in the presence of cortex only. Patches of dopaminergic fibers, revealed by tyrosine hydroxylase immunoreactivity, were observed in the majority of nigral-striatal co-cultures. Immunostaining for the AMPA-type glutamate receptor GluR1 revealed a dense patch distribution in nearly all cultures, which developed in embryonic day 19 cultures after at least six days in vitro. These findings indicate that striatal patch/matrix organization is maintained in organotypic culture, and can be induced to form in vitro in striatal slices removed from fetuses prior to the time of in vivo patch formation. Furthermore, dopaminergic innervation from co-cultured pieces of ventral mesencephalon enhances patch formation in organotypic cultures.