Parkinson’s disease is a disorder mainly characterized by progressive degeneration of dopamine producing neurons in the substantia nigra of the midbrain. The most commonly used treatment strategy is to pharmacologically restore the lost function by the administration of the dopaminergic precursor L-DOPA. Another treatment strategy is to replace the degenerated neurons with immature fetal ventral mesencephalic tissue, or ultimately stem cell-derived tissue. Grafting trials have, however, revealed poor reinnervation capacity of the grafts, leaving much of the striata dopamine-denervated. An additional drawback is the upcoming of dyskinesia (involuntary movements), a phenomenon also observed during L-DOPA treatment of Parkinson’s disease patients. Attempts to characterize nerve fiber formation from dopamine neurons have demonstrated that the nerve fibers are formed in two morphologically diverse outgrowth patterns, one early outgrowth seen in the absence of astrocytes and one later appearing outgrowth seen in co-existence with astrocytes. The overall objective of this thesis has been to study the dopaminergic outgrowth including guidance of nerve fiber formation, and to look into the mechanisms of L-DOPA-induced dyskinesia. The first paper in this thesis characterizes the different outgrowth patterns described above and their relation to different glial cells. The study demonstrated the two different outgrowth patterns to be a general phenomenon, applying not only to dopamine neurons. Attempts of characterization revealed no difference of origin in terms of dopaminergic subpopulations, i.e. A9 or A10, between the outgrowth patterns. Furthermore, the “roller-drum” technique was found optimal for studying the dual outgrowth sequences. The second and the third paper also utilized the “roller-drum” technique in order to promote both patterns of neuronal fiber formation. The effects of glial cell line-derived neurotrophic factor (GDNF) on the formation of dopamine nerve fibers, was investigated. Cultures prepared from gdnf knockout mice revealed that dopaminergic neurons survive and form nerve fiber outgrowth in the absence of GDNF. The dopaminergic nerve fibers exhibited an outgrowth pattern consistent with that previous observed in rat. GDNF was found to exert effect on the glial-associated outgrowth whereas the non-glial-associated was not affected. Astrocytic proliferation was inhibited using cytosine β-D-arabinofuranoside, resulting in reduced glial-associated outgrowth. The non-glial-associated dopaminergic outgrowth was on the other hand promoted, and was retained over longer time in culture. Furthermore, the non-glial-associated nerve fibers were found to target the fetal frontal cortex. Different developmental stages were shown to promote and affect the outgrowths differently. Taken together, these data indicate and state the importance of astrocytes and growth factors for neuronal nerve fiber formation and guidance. It also stresses the importance of fetal donor age at the time for transplantation. The fourth and fifth studies focus on L-DOPA dynamics and utilize in vivo chronoamperometry. In study four, 6-OHDA dopamine-depleted rats were exposed to chronic L-DOPA treatment and then rated as dyskinetic or non-dyskinetic. The electrochemical recordings demonstrated reduced KCl-evoked release in the intact striatum after chronic L-DOPA treatment. Time for maximal dopamine concentration after L-DOPA administration was found to be shorter in dyskinetic animals than in non-dyskinetic animals. The serotonergic nerve fiber content in the striatum was evaluated and brains from dyskinetic animals were found to exhibit significantly higher nerve fiber density compared to non-dyskinetic animals. Furthermore, the mechanisms behind the conversion of L-DOPA to dopamine in 6-OHDA dopamine-depleted rats were studied. Local administration of L-DOPA in the striatum increased the KCl-evoked dopamine release in the intact striatum. Acute application of L-DOPA resulted sometimes in a rapid conversion to dopamine, probably without vesicle packaging. This type of direct conversion is presumably occurring in non-neuronal tissue. Furthermore, KCl-evoked dopamine releases were present upon local application of L-DOPA in the dopamine-depleted striatum, suggesting that the conversion to dopamine took place elsewhere, than in dopaminergic nerve fibers. In conclusion, these studies state the importance of astrocytes for neuronal nerve fiber formation and elucidate the complexity of L-DOPA conversion in the brain.

Anxiety disorders are very common and the primary feature is abnormal or inappropriate anxiety. Fear and anxiety is often mediated by the amygdala, a brain structure rich in substance P (SP) and neurokinin 1 (NK1) receptors. To learn more about how the human amygdala is modulated by fear and anxiety in event-triggered anxiety disorders and to investigate if the SP/NK1 receptor system is affected, regional cerebral blood flow (rCBF) ([15O]-water; Study I and II) and the SP/NK1 receptor system ([11C]GR205171; Study III and IV) were studied with positron emission tomography (PET). In Study I we investigated the neural correlates of affective startle modulation in persons with specific phobia by measuring rCBF during exposure to fearful and non-fearful pictures, paired and unpaired with acoustic startle stimuli. Fear-potentiated startle was associated with activation of the affective part of the anterior cingulate cortex and the left amygdaloid–hippocampal area. In Study II short-term drug treatment effects on rCBF in patients diagnosed with social phobia was evaluated, comparing the NK1 receptor antagonist GR205171 to the selective serotonin reuptake inhibitor citalopram and placebo. Social anxiety and neural activity in the medial temporal lobe including the amygdala was significantly reduced by both drugs but not placebo. In Study III we investigated if activity in the SP/NK1 receptor system in the amygdala would be affected by fear provocation in individuals with specific snake or spider phobia. Fear provocation was associated with a decreased uptake of the NK1 antagonist [11C]GR205171 in the amygdala, possibly explained by an increase in endogenous SP release occupying the NK1 receptors. Study IV was conducted to explore the resting state NK1 receptor availability in PTSD patients as compared to healthy controls. Increased resting state binding of the tracer [11C]GR205171 in the amygdala of patients with PTSD suggested an increased amount of available receptors. In summary, fear and fear-potentiated startle modulates the human amygdala, possibly through the SP/NK1 receptor system.

A 20 weeks project at Karolinska Institutet (KI), Huddinge, Sweden is in this master thesis summarized. Alzheimer’s disease is the most common form of dementia in the world. One of the pathological hallmarks seen in AD patients consists of amyloid plaques assembled of beta amyloid (Aβ) peptide aggregates. A lot of research has been done on Aβ40 and Aβ42 but not on the longer variant with 43 residues. An earlier study by Welander et al, quantified the Aβ43 peptide from amyloid plaque cores with high-performance liquid chromatography coupled to mass-spectrometry (HPLC-MS/MS)1. Here, I present the initial development of an enzyme-linked immunosorbent assay (ELISA) with the goal to quantify Aβ43 peptides in soluble fractions of human brain tissue. An ELISA method with the possibility to quantify Aβ43 peptides from cerebral spinal fluid might have the prospect to serve as a diagnostic tool for AD in the future. Commercial ELISA kits coated with antibodies against all Aβ species was not suitable for detecting Aβ43 in soluble brain tissue from human AD patients. This is due to the high amount of Aβ40 (and in some extent Aβ42) in the samples, which will bind to the same epitope as Aβ43 on the capturing antibody. These shorter Aβ species will be in excess and bind to the capturing antibody thereby ousting Aβ43 from binding in. A better way for quantifying Aβ43 with ELISA might instead be to coat a polystyrene plate with α-Aβ43 antibodies, which are c-terminal specific to Aβ43. This will abolish the competition between the different Aβ species and function as an immunoprecipitation of unwanted species. This yielded adequate quantification of Aβ43 (2.64 pM) from tris-buffer saline (TBS) fractions from a human brain sample from AD.

Movement is central for life, and all animals depend on accurate regulation of movement for purposeful behavior. There is great diversity of movements, ranging between simple and vital breathing movements to minute and subtle movements of the face used to communicate emotions. Consequently, motor neurons, which are the only route of central nervous system output, are essential for all motor behaviors. To control the many motor behaviors expressed by an animal, motor neurons are exposed to a large number and variety of modulating synaptic inputs and have evolved into subtypes with specific functions. In this thesis, motor neuron subtypes and the synaptic input to motor neurons from Renshaw cells and Ia afferents have been studied. Novel molecular markers that identify subtypes of motor neurons are described. Three markers, Chodl, Calca and ERRβ, have been used to study the degeneration of subtypes of motor neurons in a mouse model of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Another marker, 5-ht1d, has been used to record the electrophysiological character of gamma motor neurons. In mice that lack 5-ht1d, motor neurons develop with reduced proprioceptive input. Remarkably, these mice had fewer foot faults than control animals when challenged to cross a narrow beam suggesting that the amplitude of monosynaptic proprioceptive input to motor neurons is not essential for motor coordination. In a final set of experiments, genetic removal of vesicular transport of neurotransmitter from Renshaw cells suggest that Renshaw cells are not integral for motor circuit function or motor behaviors. However, they are involved in the development of motor circuits in the spinal cord. Together, this thesis provides novel molecular tools for studies of motor neuron subtypes and novel data regarding the development and function of spinal motor circuits.

There are differences today amongst psychoanalysts regarding if psychoanalysis should limit itself to being exclusively a hermeneutic discipline or if psychoanalysis should find points of contact with neurobiology. The purpose of this essay is to touch upon the larger issue that creates the different points of view: Can psychoanalysis become enriched by finding points of contact with neurobiology, and should psychoanalysis be regarded as belonging to a broader scientific field than being exclusively a human science? The question at issue is: Can modern neurobiology contribute to a development of the psychoanalytic concepts; compulsion to repeat, transference/countertransference and talking cure? The method used is a literature study. The result shows that: Freud’s theories regarding traumatic compulsion to repeat can be linked with LeDoux’s theory of "emotional memory". Freud’s theory of the death instinct as an explanation to the compulsion to repeat can be replaced by a modern neurobiological theory of "emotional memory". The part of the countertransference that is an emotion transferred from the patient to the analyst can happen with the assistance of mirror neurons through "embodied simulation". This suggests that the phenomenon of the analyst being able to experience the patients emotion in himself does not have to imply that projection or intersubjective pressure have played a part in it. To be able to include this phenomenon in the concepts of transference and countertransference these need to be broadened or revised. It is possible to link and develop Freud’s theory of the talking cure with Deacon’s theory about symbolic communication. The results clinical implications are: Understanding of the traumatic compulsion to repeat as an expression of "emotional memory" demands work with this as an expression of memory processes. Understanding of the transference of emotion in transference/countertransference as possible through reflexive simulation processes, implies that projection or interpersonal pressure should not be preconceived in an emotion transference situation. It also implies that the emotional activation in the analyst should not by necessity be seen as an activation of the analyst’s internal objects together with emotions towards these. Deacon’s theory of symbolic communication implies that the talking cure should be used with awareness of its negative tendencies, visual thinking should not necessarily be considered non-symbolic and focus should be put on multiple ways of communication in the clinical situation.

Att ha diagnosen Aspergers syndrom innebär bl a sociala problem och psykisk sårbarhet. Denna kvalitativa studie omfattar intervjuer med fyra  psykoterapeuter och en psykolog, som har erfarenhet av att behandla personer med Aspergers syndrom. Syftet med studien är att undersöka psykoterapeuters syn på psykodynamisk psykoterapi med personer med Asperger syndrom. Frågeställningarna har varit: hur lägger man upp en terapeutisk behandling för personer med Aspergers syndrom? Vilka aspekter inom terapin med personer med Aspergers syndrom är viktiga för behandlingen?  Studien visar att teraputerna arbetar utifrån olika psykodynamiska teorier. Terapeuten behöver också ha stora kunskaper om funktionshindret. Att terapeuten har kunskap om vilka effekter funktionshindret har på personer med Asperger syndrom medför att viktiga komponenter som bemötande och arbetsallians möjliggörs och som är grunden för ett fortsatt behandlingsarbete. Vidare anses det viktigt att ta hänsyn till den specifika livssituation och historia som personer med Aspergers syndrom har, eftersom tidigare erfarenheter har betydelse för hur patienten handlar, reagerar och förstår sina livsmönster i nuet. Samtliga terapeuter betonar att personer med Aspergers syndrom i behandlingen har förbättrat sitt sociala samspel. Dessutom tycks det som att det fordras mycket av terapeuten i behandlingen med denna målgrupp eftersom det är långa behandlingar och att det är känslomässigt påfrestande att arbeta med personer med Aspergers. / To be diagnosed with Asperger's syndrome involves among other social problems and psychological vulnerability. The sample consists of four psychotherapists and one psychologist, who have experience in treating people with Asperger syndrome. The purpose of this study is to investigate the psychotherapists’ view of psychodynamic psychotherapy with people with Asperger syndrome. Questions have been; How do you set up a therapeutic treatment for people with Asperger's syndrome? What aspects in therapy with people with Asperger syndrome are important to the treatment? The study shows that therapists work from various psychodynamic theories. The therapist must also have vast knowledge of the disability. The therapist’s knowledge of the effects disability has on people with Asperger syndrome causes the key components that touch and working alliance is made possible and is the basis for continuing treatment work. Furthermore, it is important to take into account the specific situation in life and history that people with Asperger syndrome have, because past experience is relevant to how the patient react and understand their patterns of life in the present. All therapists stress that people with Asperger syndrome in treatment have improved their social interaction. Moreover, it seems that it requires a lot of the therapist in the treatment with this audience, because it is long treatments and that it is emotionally stressful to work with people with Asperger's

Post-traumatic stress disorder (PTSD) is a disabling condition that afflicts 1-10% of the general population, with twice as high lifetime prevalence for women than men. Treatments exist, but none have proven reliable and consistent efficacy. A large minority of patients remain treatment-resistant despite undergoing several different types of treatment over extended periods of time. Recently completed studies in the U.S. and in Switzerland have demonstrated the potential of 3,4-methylenedioxymethamphetamine (MDMA)-assisted psychotherapy for treatment-resistant PTSD. One of the major problems of treating PTSD is the patients’ fear state and inability to form a therapeutic alliance. Both these issues can be facilitated through administration of MDMA; the psychological effects - such as heightened empathy, increased openness and diminished anxiety – seem well-suited for therapeutic purposes. The rationale behind treating PTSD with MDMA has been indicated in neuroimaging studies; MDMA affects some of the neural structures altered in patients with PTSD, most notably the amygdala and the ventromedial prefrontal cortex. Using the Schedule 1 substance MDMA for this purpose is however controversial; animal studies have indicated that MDMA is neurotoxic, although no adverse effects on humans related to incidental use of MDMA in a controlled setting have been found. In conclusion, the data support that MDMA may be an efficient tool for treating PTSD, as well as safe and effective to use in a clinical context.

Spinal neurons are important in several aspects motor control. For example, the neurons essential for locomotor movements reside in the ventral spinal cord. In this thesis, different motor control functions are being related to neuronal populations defined by their common expression of a gene. First, a targeted disruption of the gene for vesicular glutamate transporter 2 (Vglut2/ Slc17a6) is described. The mutant animals die at birth because of their inability to breathe. The neuronal network in the brainstem, responsible for inspiration, was shown to become non-functional by the targeted deletion of Vglut2. To our surprise, it was still possible to induce rhythmic activity with normal left/right alternation in spinal cords isolated from VGLUT2-null embryos. Inconsistent reports of Vglut1 expression in the spinal cord made us re-evaluate the Vglut1 and Vglut2 expressions. While Vglut2 expression was widespread in the spinal cord, Vglut1 expression was restricted to a few cells dorsal to the central canal.  Taken together, the data suggest that, glutamatergic signaling is mandatory to drive the bilateral breathing, but not needed for coordination of basal alternating spinal locomotor rhythm. Next, a screen for genes with restricted ventral expression was made. Some of the genes found could be connected to the characteristics of specific neuronal cell populations. For example, fast motor neurons were shown to express the genes Calca and Chodl. Further, we found the Chrna2 expression selectively in putative Renshaw cells. It seems likely that the gene product, the alpha2 subunit of the nicotinergic receptor, could be linked to the unique connection of motor neurons to Renshaw cells. We used the Chrna2 promoter to drive expression of Cre recombinase in a transgenic mouse. The Cre activity was present in most neurons labeled with Renshaw cell markers, which should make it a useful tool for functional studies of this population. The studies presented here show how the genes expressed in subsets of neurons can be used to target populations of neurons for functional studies of neuronal systems.

<p>The neural code from the inner ear to the brain is dynamically controlled by central nervous efferent feedback to the audio-vestibular epithelium. Although such efference provides the basis for a cognitive control of our hearing and balance, we know surprisingly little about this feedback system. This project has investigated the applicability of a transgenic mouse model, expressing a fluorescent protein under the choline-acetyltransferase (ChAT) promoter, for targeting the cholinergic audio-vestibular efferent neurons in the brainstem. It was found that the mouse model is useful for targeting the vestibular efferents, which are fluorescent, but not the auditory efferents, which are not highlighted. This model enables, for the first time, physiological studies of the vestibular efferent neurons and their synaptic inputs. We next assessed the expression of the potassium channel family Kv4, known to generate transient potassium currents upon depolarization. Such potassium currents are found in auditory efferent neurons, but it is not known whether Kv4 subunits are expressed in these neurons. Moreover, it is not known if Kv4 is present and has a function in the vestibular efferent neurons. Double labelling with anti-ChAT and anti-Kv4.2 or Kv4.3 demonstrates that the Kv4.3 subunits are abundantly expressed in audio-vestibular efferents, thus indicating that this subunit is a large contributor to the excitability and firing properties of the auditory efferent neurons, and most probably also for the vestibular efferent neurons. In addition, we also unexpectedly found a strong expression of Kv4.3 in principal cells of the superior olive, the neurons which are important for sound localization.</p>

Artificial divergent selection of chickens for high and low body weight at 8 weeks of age has produced two lines: the high (HWS) and low (LWS) body weight chicken lines. In addition to the difference in body weight, the lines show extreme differences in feeding behaviour and body composition. The aim of this study was to uncover the genetic and molecular factors that contribute to and determine these differences, especially regarding body energy regulation and appetite. In papers I and II, genome-wide gene expression in a brain sample containing hypothalamus and in dissected hypothalamus was analysed using DNA microarray and qRT-PCR. We found that levels of differential expression were generally moderate, which was consistent with the idea that polygenic factors were involved in the establishment of the chicken lines. Genes associated with neural plasticity, lipid metabolism and body energy regulation were differentially expressed. This result indicated that the neural systems regulating feeding behaviour and body weight were altered in the chicken lines. However, genes that were involved in the central melanocortin system were not systematically differentially expressed. Interestingly, the biggest differences in expression between the lines found in endogenous retrovirus sequences of the ALV subgroup E. Thus, in paper III, we characterized the number of integrations, the expression of ALVE retroviral elements and their effects on body weight. A significant correlation between low body weight and high ALVE expression was observed in female F9 birds from an HWS x LWS advanced intercross line. This implied that ev-loci contributing to increased ALVE expression levels were genetically linked to loci influencing the low body weight of the pullets. In paper IV, the carnitine palmitoyltransferase-1b gene (CPT1B), which was highly differentially expressed in the hypothalami, was investigated. We mapped chicken CPT1B to the distal tip of chromosome 1p. The levels of CPT1B mRNA in the HWS line were higher in the hypothalamus and lower in muscle than in the LWS line. This pattern of differential expression indicates that this gene could contribute to the remarkable phenotypic differences between HWS and LWS chickens. However, comparison with quantitative trait loci data showed that the expression of CPT1B is a trans effect, rather than a direct causative locus. In conclusion, the data suggested that the long-term selection for body weight resulted in differential gene expression in the brains of the selected chicken lines. These results may have relevance for the poultry industry and will also contribute to increasing knowledge about human diseases such as obesity and anorexia.