Sara af Bjerkén

HISTOLOGICAL STUDIES OF THE EFFECTS OF CHRONIC IMPLANTATION OF CERAMIC-BASED MICROELECTRODE ARRAYS AND MICRODIALYSIS PROBES IN RAT PREFRONTAL CORTEX

Chronic implantation of neurotransmitter measuring devices is essential for awake, behavioral studies occurring over multiple days. Little is known regarding the effects of long term implantation on surrounding brain parenchyma and the resulting alterations in the functional properties of this tissue. We examined the extent of tissue damage produced by chronic implantation of either ceramic microelectrode arrays (MEAs) or microdialysis probes. Histological studies were carried out on fixed tissues using stains for neurons (cresyl violet), astrocytes (GFAP), microglia (Iba1), glutamatergic nerve fibers (VGLUT1), and the blood-brain barrier (SMI-71). Nissl staining showed pronounced tissue body loss with microdialysis implants compared to MEAs. The MEAs produced mild gliosis extending 50-100 microm from the tracks, with a significant change in the affected areas starting at 3 days. By contrast, the microdialysis probes produced gliosis extending 200-300 microm from the track, which was significant at 3 and 7 days. Markers for microglia and glutamatergic fibers supported that the MEAs produce minimal damage with significant changes occurring only at 3 and 7 days that return to control levels by 1 month. SMI-71 staining supported the integrity of the blood-brain barrier out to 1 week for both the microdialysis probes and the MEAs. This data support that the ceramic MEAs small size and biocompatibility are necessary to accurately measure neurotransmitter levels in the intact brain. The minimal invasiveness of the MEAs reduce tissue loss, allowing for long term (>6 month) electrochemical and electrophysiological monitoring of brain activity.

Dopamine release from serotonergic nerve fibers is reduced in L-DOPA-induced dyskinesia.

J. Neurochem. (2011) 10.1111/j.1471‐4159.2011.07292.x

Chronic intermittent L-DOPA treatment induces changes in dopamine release

3,4‐Dihydroxyphenyl‐l‐alanine (l‐DOPA)‐induced dyskinesia often develops as a side effect of chronic l‐DOPA therapy. This study was undertaken to investigate dopamine (DA) release upon l‐DOPA treatment. Chronoamperometric measurements were performed in unilaterally DA‐depleted rats, chronically treated with l‐DOPA, resulting in dyskinetic and non‐dyskinetic animals. Normal and lesioned l‐DOPA naïve animals were used as controls. Potassium‐evoked DA releases were significantly reduced in intact sides of animals undertaken chronic l‐DOPA treatment, independent on dyskinetic behavior. Acute l‐DOPA further attenuated the amplitude of the DA release in the control sides. In DA‐depleted striata, no difference was found in potassium‐evoked DA releases, and acute l‐DOPA did not affect the amplitude. While immunoreactivity to serotonin uptake transporter was higher in lesioned striata of animals displaying dyskinetic behavior, no correlation could be documented between serotonin transporter‐positive nerve fiber density and the amplitude of released DA. In conclusions, the amplitude of potassium‐evoked DA release is attenuated in intact striatum after chronic intermittent l‐DOPA treatment. No change in amplitude was found in DA‐denervated sides of either dyskinetic or non‐dyskinetic animals, while release kinetics were changed. This indicates the importance of studying DA release dynamics for the understanding of both beneficial and adverse effects of l‐DOPA replacement therapy.

Glial influence on nerve fiber formation from rat ventral mesencephalic organotypic tissue cultures

Rat fetal ventral mesencephalic organotypic cultures have demonstrated two morphologically different dopamine nerve fiber growth patterns, in which the initial nerve fibers are formed in the absence of astrocytes and the second wave is guided by astrocytes. In this study, the presence of subpopulations of dopamine neurons, other neuronal populations, and glial cells was determined. We used “roller‐drum” organotypic cultures, and the results revealed that β‐tubulin‐positive/tyrosine hydroxylase (TH)‐negative nerve fibers were present as early as 1 day in vitro (DIV). A similar growth pattern produced by TH‐positive neurons was present from 2 DIV. These neurites grew to reach distances over 4 mm and over time appeared to be degenerating. Thin, vimentin‐positive processes were found among these nerve fibers. As the first growth was retracted, a second outgrowth was initiated and formed on migrating astrocytes. TH‐ and aldehyde dehydrogenase‐1 (ALDH1)‐positive nerve fibers formed both the nonglia‐associated and the glia‐associated outgrowth. In cultures with membrane inserts, only the glia‐associated outgrowth was found. Vimentin‐positive cells preceded migration of NG2‐positive oligodendrocytes and Iba‐1‐positive microglia. Oligodendrocytes appeared not to be involved in guiding neuritic growth, but microglia was absent over areas dense with TH‐positive neurons. In conclusion, in “roller‐drum” cultures, nerve fibers are generally formed in two sequences. The early‐formed nerve fibers grow in the presence of thin, vimentin‐positive processes. The second nerve fiber outgrowth is formed on astroglia, with no correlation to the presence of oligodendrocytes or microglia. ALDH1‐positive nerve fibers, presumably derived from A9 dopamine neurons, participate in formation of both sequences of outgrowth. J. Comp. Neurol. 501:431–442, 2007.

Effects of glial cell line-derived neurotrophic factor deletion on ventral mesencephalic organotypic tissue cultures

Glial cell line-derived neurotrophic factor (GDNF) is potent for survival and promotion of nerve fibers from midbrain dopamine neurons. It is also known to exert different effects on specific subpopulations of dopamine neurons. In organotypic tissue cultures, dopamine neurons form two diverse nerve fiber growth patterns, targeting the striatum differently. The aim of this study was to investigate the effect of GDNF on the formation of dopamine nerve fibers. Organotypic tissue cultures of ventral mesencephalon of gdnf gene-deleted mice were studied. The results revealed that dopamine neurons survive in the absence of GDNF. Tyrosine hydroxylase immunoreactivity demonstrated, in gdnf knockout and wildtype cultures, nerve fiber formation with two separate morphologies occurring either in the absence or the presence of astrocytes. The outgrowth that occurred in the absence of astrocytes was unaffected by gdnf deletion, whereas nerve fibers guided by the presence of astrocytes were affected in that they reached significantly shorter distances from the gdnf gene-deleted tissue slice, compared to those measured in wildtype cultures. Treatment with GDNF reversed this effect and increased nerve fiber density independent of genotype. Furthermore, migration of astrocytes reached significantly shorter distances from the tissue slice in GDNF knockout compared to wildtype cultures. Exogenous GDNF increased astrocytic migration in gdnf gene-deleted tissue cultures, comparable to lengths observed in wildtype tissue cultures. In conclusion, cultured midbrain dopamine neurons survive in the absence of GDNF, and the addition of GDNF improved dopamine nerve fiber formation - possibly as an indirect effect of astrocytic stimulation.

Inhibition of astrocytes promotes long-distance growing nerve fibers in ventral mesencephalic cultures

Tyrosine hydroxylase‐positive nerve fiber formation occurs in two diverse morphological patterns in rat fetal ventral mesencephalic slice cultures; one is non‐glial‐associated and the other is glial‐associated. The aim of this study was to characterize the non‐glial‐associated nerve fibers and its relation to migration of astrocytes. Organotypic slice cultures were prepared from embryonic days 12, 14, and 18 rat fetuses and maintained for 5, 7 or 14 days in vitro. Inhibition of cell proliferation using cytosine β‐d‐arabinofuranoside was conducted in embryonic day 14 ventral mesencephalic cultures. The treatment impaired astrocytic migration at 7 and 14 days in vitro. The reduced migration of astrocytes exerted a negative effect on the glial‐associated tyrosine hydroxylase‐positive nerve fibers, reducing the outgrowth from the tissue slice. The non‐glial‐associated outgrowth was, however, positively affected by reduced astrocytic migration, reaching distances around 3 mm in 2 weeks, and remained for longer time in culture. Co‐cultures of fetal ventral mesencephalon and frontal cortex revealed the cortex as a target for the non‐glial‐associated tyrosine hydroxylase‐positive outgrowth. The age of the fetal tissue at plating affected the astrocytes such that older tissue increased the length of astrocytc migration. Younger tissue at plating promoted the presence of non‐glial‐asscociated outgrowth and long radial‐glia‐like processes, while older tissue promoted migration of neurons instead of formation of nerve fiber network. In conclusion, inhibition of astrocytic proliferation promotes the persistence of long‐distance growing tyrosine hydroxylase‐positive nerve fibers in ventral mesencephalic slices cultures. Furthermore, the long‐distance growing nerve fibers target the frontal cortex and are absent in cultures derived from older tissue.

Degradation of proteoglycans affects astrocytes and neurite formation in organotypic tissue cultures.

Chondroitin sulfate proteoglycans (CSPGs) promote nerve growth during development, and inhibit axonal growth in the adult CNS after injury. Chondroitinase ABC (ChABC) and methyl-umbelliferyl-β-d-xyloside (β-xyloside), two compounds that degrade CSPGs, promote regeneration after injury, however, they demonstrate opposing results in tissue culture. To elucidate the effect of the two compounds, organotypic tissue cultures, treated with ChABC or β-xyloside, were employed to monitor nerve fiber outgrowth and astrocytic migration. Rat ventral mesencephalon (VM) and spinal cord (SC) from embryonic day (E) 14 and E18 were treated early, from the plating day for 14 days in vitro, or late where treatment was initiated after being cultured for 14 days. In the early treatment of E14 VM and SC cultures, astrocytic migration and nerve fiber outgrowth were hampered using both compounds. Early treatment of E18 cultures reduced the astrocytic migration, while nerve growth was promoted by β-xyloside, but not by ChABC. In the late treated cultures of both E14 and E18 cultures, no differences in distances that astrocytes migrated or nerve fiber growth were observed. However, in β-xyloside-treated cultures, the confluency of astrocytic monolayer was disrupted. In E18 cultures both early and late treatments, neuronal migration was present in control cultures, which was preserved using ChABC but not β-xyloside. In conclusion, ChABC and β-xyloside had similar effects and hampered nerve fiber growth and astrocytic migration in E14 cultures. In E18 cultures nerve fiber growth was stimulated and neuronal migration was hampered after β-xyloside treatment while ChABC treatment did not exert these effects.

An altered blood–brain barrier contributes to brain iron accumulation and neuroinflammation in the 6-OHDA rat model of Parkinson’s disease

Brain iron accumulation is a common feature shared by several neurodegenerative disorders including Parkinsons disease. However, what produces this accumulation of iron is still unknown. In this study, the 6-hydroxydopamine (6-OHDA) hemi-parkinsonian rat model was used to investigate abnormal iron accumulation in substantia nigra. We investigated three possible causes of iron accumulation; a compromised blood-brain barrier (BBB), abnormal expression of ferritin, and neuroinflammation. We identified alterations in the BBB subsequent to the injection of 6-OHDA using gadolinium-enhanced magnetic resonance imaging (MRI). Moreover, detection of extravasated IgG suggested that peripheral components are able to enter the brain through a leaky BBB. Presence of iron following dopamine cell degeneration was studied by MRI, which revealed hypointense signals in the substantia nigra. The presence of iron deposits was further validated in histological evaluations. Furthermore, iron inclusions were closely associated with active microglia and with increased levels of L-ferritin indicating a putative role for microglia and L-ferritin in brain iron accumulation and dopamine neurodegeneration.

Dampened amphetamine-stimulated behavior and altered dopamine transporter function in the absence of brain GDNF

Midbrain dopamine neuron dysfunction contributes to various psychiatric and neurological diseases, including drug addiction and Parkinsons disease. Because of its well established dopaminotrophic effects, the therapeutic potential of glial cell line-derived neurotrophic factor (GDNF) has been studied extensively in various disorders with disturbed dopamine homeostasis. However, the outcomes from preclinical and clinical studies vary, highlighting a need for a better understanding of the physiological role of GDNF on striatal dopaminergic function. Nevertheless, the current lack of appropriate animal models has limited this understanding. Therefore, we have generated novel mouse models to study conditional Gdnf deletion in the CNS during embryonic development and reduction of striatal GDNF levels in adult mice via AAV-Cre delivery. We found that both of these mice have reduced amphetamine-induced locomotor response and striatal dopamine efflux. Embryonic GDNF deletion in the CNS did not affect striatal dopamine levels or dopamine release, but dopamine reuptake was increased due to increased levels of both total and synaptic membrane-associated dopamine transporters. Collectively, these results suggest that endogenous GDNF plays an important role in regulating the function of dopamine transporters in the striatum. SIGNIFICANCE STATEMENT Delivery of ectopic glial cell line-derived neurotrophic factor (GDNF) promotes the function, plasticity, and survival of midbrain dopaminergic neurons, the dysfunction of which contributes to various neurological and psychiatric diseases. However, how the deletion or reduction of GDNF in the CNS affects the function of dopaminergic neurons has remained unknown. Using conditional Gdnf knock-out mice, we found that endogenous GDNF affects striatal dopamine homeostasis and regulates amphetamine-induced behaviors by regulating the level and function of dopamine transporters. These data regarding the physiological role of GDNF are relevant in the context of neurological and neurodegenerative diseases that involve changes in dopamine transporter function.