Differential nuclear localization of complexes may underlie in vivo intrabody efficacy in Huntington's disease.

Intrabodies offer attractive options for manipulating the protein misfolding that triggers neurodegenerative diseases. In Huntington's disease, where the expanded polyglutamine tract in the extreme N-terminal region of huntingtin exon1 misfolds, two lead intrabodies have been selected against an adjacent peptide, using slightly different approaches. Both are effective at preventing aggregation of a reporter fragment in transient co-transfection assays. However, after intracranial delivery to mutant mouse brains, VL12.3, which is mainly localized to the nucleus, appears to accelerate the mutant phenotype, while C4 scFv, which is largely cytoplasmic, shows partial phenotypic correction. This comparison highlights parameters that could inform intrabody therapeutics for multiple proteostatic diseases.

Spatiotemporal analysis of Fos expression associated with cocaine- and PTZ-induced seizures in prenatally cocaine-treated rats.

We previously reported that prenatal cocaine exposure (40 mg/kg s.c., E10-E20) increased susceptibility to convulsant-induced seizures later in life, with female rats becoming more sensitive to seizures induced by cocaine and pentylenetetrazol (PTZ), and males more sensitive to PTZ-induced seizures (Snyder-Keller and Keller, 1995, 2000). In order to determine the locus of enhanced seizure susceptibility in the brains of prenatally cocaine-treated rats, we examined the distribution and density of Fos-immunoreactive cells after cocaine- and PTZ-induced seizures in mature rats. Subconvulsive cocaine doses induced c-fos in cortical areas as well as densely dopamine-innervated regions such as striatum and nucleus accumbens. Following cocaine-induced seizures, intense c-fos induction was observed in piriform cortex, amygdala, and hippocampus. Quantification of the number of Fos-immunoreactive cells in the brains of prenatally cocaine-treated versus prenatally saline-treated rats revealed differences in piriform cortex and amygdala that were indicative of a lower threshold in prenatally cocaine-treated female rats. Following PTZ-induced seizures, the same pattern of limbic structures were recruited with increasing seizure severity. Only females exhibited changes in the number of Fos-immunoreactive cells as a result of prenatal cocaine treatment. Pretreatment with the noncompetitive NMDA antagonist MK-801 blocked both cocaine- and PTZ-induced seizures, and Fos expression in limbic areas was also blocked. The dopamine D1 antagonist SCH 23390 blocked cocaine-induced seizures and associated c-fos induction, but not PTZ-induced seizures or Fos. Examination of the pattern of Fos expression at 15-20 min postseizure revealed that the initial site of c-fos induction associated with PTZ-induced seizures appeared to be the piriform cortex, whereas cocaine-induced seizures induced early expression in both piriform cortex and lateral amygdala. These findings suggest that neural alterations residing in the piriform cortex and amygdala are likely to account for the increased seizure susceptibility of prenatally cocaine-treated rats.

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 Dulbecco's modified Eagle's 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.

Prenatal cocaine exposure.

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 newborns 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, Parkinson's disease, etc.

Enhanced susceptibility to cocaine- and pentylenetetrazol-induced seizures in prenatally cocaine-treated rats.

We previously reported that prenatal cocaine exposure increased susceptibility to cocaine-induced seizures later in life. Here we examine whether this enhanced susceptibility to seizures generalizes to other chemoconvulsants, and whether postnatal cocaine treatment similarly increases susceptibility. Following prenatal cocaine treatment (40 mg/kg; E10-20), both male and female rats were more likely to seize to a dose of 30 mg/kg pentylenetetrazol (PTZ) at 2 months of age, although the severity of the seizures observed was increased only in females. Daily cocaine injections (10-20 mg/kg SC) during the first 10 days after birth also produced effects that were dependent on the sex of the animal. Postnatally cocaine-treated female rats showed no greater incidence of seizures in response to an acute high dose of cocaine, but did exhibit an increased susceptibility to cocaine-kindled seizures. Male, but not female, postnatally cocaine-treated rats were more susceptible to PTZ-induced seizures. The increased susceptibility to seizures induced by two different chemoconvulsants after prenatal cocaine treatment suggests that developmental cocaine exposure, particularly during the second trimester equivalent, alters the balance between excitation and inhibition in the brain.

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.

Stimulant-mediated c-fos induction in striatum as a function of age, sex, and prenatal cocaine exposure.

Induction of the immediate-early gene c-fos by the stimulants cocaine and amphetamine (AMPH) was analyzed by Fos immunocytochemistry at different ages in the brains of prenatally cocaine-treated and control rats. Cocaine and AMPH induced c-fos in patches of striatal neurons during the first postnatal week, and thereafter produced a progressively more homogeneous pattern that was more dense medially. Quantification of Fos-immunoreactive cells in older rats revealed differences related to sex and prenatal cocaine treatment. Both cocaine and AMPH produced dose-dependent increases in the number of Fos-immunoreactive cells in striatum. Prenatal cocaine exposure resulted in increased Fos in males in response to AMPH (2 mg/kg) at P18 and cocaine (10 mg/kg) at 1-2 months. In females, prenatal cocaine treatment resulted in a reduced response to cocaine at 1-2 months. Increased c-fos induction was observed in control females compared to control males in response to low doses of stimulants; no such sex difference was observed in prenatally cocaine-treated rats. The dopamine D1 antagonist SCH23390 blocked cocaine-mediated c-fos induction in all groups. The NMDA antagonist MK-801 blocked cocaine-mediated c-fos induction in the medial striatum. In females only, MK-801 pretreatment resulted in a dramatic increase in the number of Fos-immunoreactive cells in lateral striatum. These findings indicate differences in the neural basis of c-fos induction in males and females, and changes in stimulant-mediated c-fos induction resulting from prenatal cocaine exposure.

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.

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.

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.

Reciprocal influences of nigral cells and striatal patch neurons in dissociated co-cultures.

Our previous work has shown that the functional efficacy of nigral tissue transplants into dopamine (DA)-depleted rats is increased when embryonic striatal tissue is included (Costantini et al.: Exp Neurol 127:219-231, 1994). To examine further the influence of striatal patch neurons in this regard, we employed co-cultures of dissociated nigral and striatal cells taken from embryos at different ages. Striatal patch neurons were labeled by in vivo bromodeoxyuridine (BrdU) on embryonic day (E)13 and E14. The percentage of striatal cells that were BrdU labeled was greater in E14 striatal cultures (51.0%) compared with E16 (33.9%) and E20 (3.5%) striatal cultures at 1 day in vitro (DIV). The proportion of surviving BrdU-labeled cells in striatal cultures decreased over time. The inclusion of E14 nigral cells attenuated this decline. Similarly, the number of dopaminergic [tyrosine hydroxylase (TH)-immunoreactive] neurons in pure nigral cultures decreased with time in vitro (8.2% at 1 DIV to 3.5% at 12-15 DIV). The inclusion of E14 striatal tissue increased the number of TH-immunoreactive neurons at all time points, whereas E16 and E20 striatal tissue was somewhat less effective. Thus, the survival of nigral DA neurons and striatal patch neurons in culture appears to be enhanced in the presence of the other. These reciprocal influences on neuronal survival may be relevant to the in vivo development of the nigrostriatal system as well as the enhanced function of cells in co-transplants.

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.

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.

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.

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.

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.

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.