Ville Sallinen

The comparative neuroanatomy and neurochemistry of zebrafish CNS systems of relevance to human neuropsychiatric diseases.

Modulatory neurotransmitters which signal through G protein-coupled receptors control brain functions which deteriorate in degenerative brain diseases. During the past decade many of these systems have been mapped in the zebrafish brain. The main architecture of the systems in zebrafish brain resembles that of the mammals, despite differences in the development of the telencephalon and mesodiencephalon. Modulatory neurotransmitters systems which degenerate in human diseases include dopamine, noradrenaline, serotonin, histamine, acetylcholine and orexin/hypocretin. Although the number of G protein-coupled receptors in zebrafish is clearly larger than in mammals, many receptors have similar expression patterns, binding and signaling properties as in mammals. Distinct differences between mammals and zebrafish include duplication of the tyrosine hydroxylase gene in zebrafish, and presence of one instead of two monoamine oxidase genes. Zebrafish are sensitive to neurotoxins including MPTP, and exposure to this neurotoxin induces a decline in dopamine content and number of detectable tyrosine hydroxylase immunoreactive neurons in distinct nuclei. Sensitivity to important neurotoxins, many available genetic methods, rapid development and large-scale quantitative behavioral methods in addition to advanced quantitative anatomical methods render zebrafish an optimal organism for studies on disease mechanisms.

Modulatory Neurotransmitter Systems and Behavior: Towards Zebrafish Models of Neurodegenerative Diseases

The modulatory aminergic neurotransmitters are involved in practically all important physiological systems in the brain, and many of them are also involved in human central nervous system diseases, including Parkinsons disease, schizophrenia, Alzheimers disease, and depression. The zebrafish brain aminergic systems share many structural properties with the mammalian systems. The noradrenergic, serotonergic, and histaminergic systems are highly similar. The dopaminergic systems also show similarities with the major difference being the lack of dopaminergic neurons in zebrafish mesencephalon. Development of automated quantitative behavioral analysis methods for zebrafish and imaging systems of complete brain neurotransmitter networks have enabled comprehensive studies on these systems in normal and pathological conditions. It is possible to visualize complete neurotransmitter systems in the whole zebrafish brain at an age when the fish already displays all major vital behaviors except reproduction. Alterations of brain dopaminergic systems with MPTP, the neurotoxin that in humans and rodents induces Parkinsons disease, induces both changes in zebrafish dopaminergic system and quantifiable abnormalities in motor behavior. Chemically-induced brain histamine deficiency causes an identifiable alteration in histaminergic neurons and terminal networks, and a clear change in swimming behavior and long-term memory. Combining the imaging techniques and behavioral methods with zebrafish genetics is likely to help reveal how the modulatory transmitter systems interact to produce important behaviors, and how they are regulated in pathophysiological conditions and diseases.

Hyperserotonergic phenotype after monoamine oxidase inhibition in larval zebrafish

Serotonin (or 5‐hydroxytryptamine; 5‐HT) and monoamine oxidase (MAO) are involved in several physiological functions and pathological conditions. We show that the serotonergic system and its development in zebrafish are similar to those of other vertebrates rendering zebrafish a good model to study them. Development of MAO expression followed a similar time course as the 5‐HT system. MAO expression and activity were located in or adjacent to serotonergic nuclei and their targets, especially in the ventral hypothalamus. MAO mRNA was detected in the brain from 24 h post‐fertilization and histochemical enzyme activity from 42 h post‐fertilization. Deprenyl (100 μM) decreased MAO activity 34–74% depending on the age. Inhibition of MAO by deprenyl strongly increased 5‐HT but not dopamine and noradrenaline levels. Deprenyl decreased 5‐HT‐immunoreactivity in serotonergic neurons and induced novel ectopic 5‐HT‐immunoreactivity neurons in the diencephalon in a manner dependent on 5‐HT uptake. Deprenyl administration decreased locomotion, altered vertical positioning and increased heart rate. Treatment with p‐chlorophenylalanine normalized 5‐HT levels and rescued the behavioral alteration, indicating that the symptoms were 5‐HT dependent. These findings suggest that zebrafish MAO resembles mammalian MAO A more than MAO B, metabolizing mainly 5‐HT. Applications of this model of hyperserotonergism include pharmacological and genetic screenings.

Distinct structure and activity of monoamine oxidase in the brain of zebrafish (Danio rerio).

Monoamine oxidase (MAO) is a mitochondrial flavoprotein involved in the metabolism of, e.g., aminergic neurotransmitters and the parkinsonism‐inducing neurotoxin 1‐methyl‐4‐phenyl‐1,2,5,6‐tetrahydropyridine (MPTP). We have reported earlier MPTP‐related alterations of brain catecholaminergic system in zebrafish (Danio rerio) brain. Here we describe the structural and functional properties of zebrafish MAO and the distribution of MAO mRNA and activity in zebrafish brain. The gene is located in chromosome 9 and consists of 15 exons. The amino acid composition of the active center resembles both human MAO‐A and MAO‐B. The enzyme displayed the highest substrate specificity for tyramine, followed by serotonin, phenylethylamine, MPTP, and dopamine; isoform‐specific antagonists blocked the activity of the enzyme with equal potency. Zebrafish MAO mRNA, which was present in several tissues, and enzyme displayed differential distribution in the brain; dopaminergic cell clusters had low to moderate levels of MAO activity, whereas the highest levels of MAO activity were detected in noradrenergic and serotonergic cell groups and the habenulointerpeduncular pathway, including its caudal projection to the medial ventral rhombencephalon. The results of this study confirm the presence of functionally active MAO in zebrafish brain and other tissues and characterize the neural systems that express MAO and areas of intense activity in the brain. They also suggest that MPTP toxicity not related to MAO may affect the zebrafish brain. J. Comp. Neurol. 498:593–610, 2006.

Dopaminergic cell damage and vulnerability to MPTP in Pink1 knockdown zebrafish

The functions of PTEN (phosphatase/tensin homolog)-induced putative kinase (PINK1), which is mutated in early-onset Parkinsons disease, are poorly understood. We characterized a PINK1 antibody and found colocalization of PINK1-like immunoreactivity with aminergic markers. We inactivated translation of Pink1 using morpholino-oligonucleotides (MOs) in larval zebrafish. Dopaminergic neurons consisted of two sets of neuron populations, marked by complementary expression of two tyrosine hydroxylase genes th1 and th2. Translation inhibition of pink1 resulted in reduction of both th mRNA forms until day 5 or 7, respectively. The affected dopaminergic neurons were in one group expressing th1 and three groups expressing th2. Lack of Pink1 sensitized the fish to subeffective doses of MPTP, which caused a locomotor deficit and facilitated loss of th1 in one diencephalic dopaminergic cell group. Control experiments with pink1 mRNA and control MO suggested that effects with the splice site targeting MO were specific. Distinct groups of dopaminergic neurons are thus sensitive to loss of Pink1. Sensitization of the pink1 morphant fish to MPTP toxicity suggests that genetic factors play a role in toxin-induced Parkinsons disease.

Nonoperative management of perforated diverticulitis with extraluminal air is safe and effective in selected patients.

BACKGROUND: The optimal treatment for diverticulitis with extraluminal air is controversial. OBJECTIVE: The purpose of this research was to evaluate the safety and effectiveness of nonoperative treatment of acute diverticulitis with extraluminal air. DESIGN: This was a retrospective cohort. SETTINGS: The study was conducted at an academic teaching hospital functioning as both a tertiary and secondary care referral center. PATIENTS: All of the patients with CT-diagnosed acute perforated diverticulitis with extraluminal air from 2006 through 2010 were included in this study. INTERVENTIONS: Nonoperative treatment composed of intravenous antibiotics, bowel rest, and percutaneous drainage were the included interventions. MAIN OUTCOME MEASURES: The need for operative management and mortality were measured. RESULTS: A total of 132 patients underwent nonoperative treatment, whereas 48 patients were primarily operated on. Patients treated nonoperatively were divided into 3 groups on the basis of identified factors that independently predicted risk for failure: 1) patients with pericolic air (n = 82) without abscess had a 99% success rate with 0% mortality. 2) Patients with distant intraperitoneal air (n = 29) had a 62% success ratewith 0% mortality. Abundant distant intraperitoneal air and fluid in the fossa Douglas were identified as risk factors for failure. Patients without these risk factors had an 86% success rate with nonoperative management. 3) Patients with distant retroperitoneal air (n = 14) had a 43% success rate with 7% mortality. LIMITATIONS: Comparison of nonoperative versus operative treatment cannot be made because of the study’s retrospective nature. CONCLUSIONS: Nonoperative treatment of acute diverticulitis with extraluminal air is safe and effective in patients with a small amount of distant intraperitoneal air or pericolic air without clinical signs of peritonitis.

Symptomatic Treatment for Uncomplicated Acute Diverticulitis: A Prospective Cohort Study

BACKGROUND: Even though evidence for nonantibiotic treatment of uncomplicated diverticulitis exists, it has not gained widespread adoption. OBJECTIVE: The aim of this prospective single-arm study was to analyze the safety and efficacy of symptomatic (nonantibiotic) treatment for uncomplicated diverticulitis during a 30-day follow-up period. DESIGN: This study is a single-arm prospective trial (ClinicalTrials.gov ID NCT02219698). SETTINGS: This study was performed at an academic teaching hospital functioning as both a tertiary and secondary care referral center. PATIENTS: Patients, who had CT-verified uncomplicated acute colonic diverticulitis (including diverticulitis with pericolic air), were evaluated for the study. Patients with ongoing antibiotic therapy, immunosuppression, suspicion of peritonitis, organ dysfunction, pregnancy, or other infections requiring antibiotics were excluded. INTERVENTIONS: Symptomatic in- or outpatient treatment consisted of mild analgesics (nonsteroidal anti-inflammatory drug or paracetamol). MAIN OUTCOME MEASURES: The incidence of complicated diverticulitis was the primary outcome. RESULTS: Overall, 161 patients were included in the study, and 153 (95%) completed the 30-day follow-up. Four (3%) of these patients were misdiagnosed (abscess in the initial CT scan). A total of 14 (9%) patients had pericolic air. Altogether, 140 (87%) patients were treated as outpatients, and 4 (3%) of them were admitted to the hospital during the follow-up. None of the patients developed complicated diverticulitis or required surgery, but, 2 days (median) after inclusion, antibiotics were given to 14 (9%, 6 orally, 8 intravenously) patients. LIMITATIONS: This study is limited by the lack of a comparison group and by the relatively short follow-up. CONCLUSIONS: Symptomatic treatment of uncomplicated diverticulitis without antibiotics is safe and effective.

Outcomes of resected nonfunctional pancreatic neuroendocrine tumors: Do size and symptoms matter?

BACKGROUND Nonfunctional pancreatic neuroendocrine tumors (NF-PNETs) are rare tumors with highly variable outcome. Current guidelines recommend surveillance for small tumors (≤ 2 cm), but a scientific basis for such recommendation is scarce. METHODS Patients who underwent surgery for NF-PNET during 2001-2013 were identified from a prospectively maintained database and reviewed retrospectively. RESULTS Fifty-eight patients that had undergone an operative procedure for NF-PNET were identified. Forty-one patients (71%) were symptomatic. Median size of the tumor was 2.5 cm (range 0.9-12.0 cm). WHO 2010 grade was predictive of both overall- and disease-free survival (P < .001), whereas size alone was not. Twenty-four patients had a small NF-PNET (≤ 2 cm), of whom 16 were symptomatic and 8 asymptomatic. Seven patients with small symptomatic NF-PNETs showed signs of malignant behavior: 4 had lymph node metastases, 1 had liver metastases before surgery, 3 developed liver metastases, and 3 died of the disease. All 7 patients had either bile duct or pancreatic duct obstruction or both on preoperative imaging. On the contrary, patients with small asymptomatic NF-PNETs did not develop distant metastases nor died of disease. CONCLUSION The 2010 grading system from the World Health Organization can be used to predict survival. Symptomatic small NF-PNETs that caused bile and/or pancreatic duct obstruction had poor outcome. In contrast, asymptomatic small NF-PNETs seem to have benign course, and are candidates for surveillance.

Galanin gene expression and effects of its knock-down on the development of the nervous system in larval zebrafish

Despite the known importance of galanin in the nervous system of vertebrates, the galanin gene structure and expression and the consequences of galanin deficiency in developing zebrafish are unknown. We cloned the galanin gene and analyzed its expression by using in situ hybridization, PCR, and immunocytochemistry throughout the early development of zebrafish until the end of the first week of life. The single zebrafish galanin gene encoded for a single amidated galanin peptide and a galanin message‐associated peptide. Two forms resulting from alternative processing were identified. Galanin mRNA was maternally expressed and found in developing fish throughout early development. In situ hybridization showed the first positive neurons in three groups in the brain at 28 hours postfertilization. At 2 days postfertilization, three prosencephalic neuron groups were seen in the preoptic area and in rostral and caudal periventricular hypothalamus. In addition, two other groups of weakly stained neurons were visible, one in the midbrain and another in the hindbrain. Translation inhibition of galanin mRNA with morpholino oligonucleotides caused complete disappearance of galanin immunoreactivity in the brain until 7 dpf and did not induce known cascades of nonspecific pathways or morphological abnormalities. A minor disturbance of sensory ganglia was found. Galanin knockdown did not alter the expression of tyrosine hydroxylases 1 and 2, choline acetyltransferase, histidine decarboxylase, or orexin mRNA. The results suggest that galanin does not regulate the development of these key markers of specific neurons, although galanin‐expressing fibers were in a close spatial proximity to several neurons of these neuronal populations. J. Comp. Neurol. 520:3846–3862, 2012.

Staging of acute diverticulitis based on clinical, radiologic, and physiologic parameters.

BACKGROUND Acute diverticulitis is a broad spectrum of diseases with highly varying mortality and need for surgery. The aim of this study was to create an accurate staging of diverticulitis, which could be used both preoperatively and intraoperatively to predict outcome and guide treatment. METHODS This was a retrospective study of patients treated for diverticulitis in a secondary and tertiary referral center. Multivariate analysis was performed on several clinical, radiologic, and physiologic parameters to find predictors of mortality, need for surgery, need for intensive care, and length of stay. RESULTS A total of 631 patients were analyzed. Organ dysfunction, peritonitis, and abscess size greater than 6 cm were identified as independent predictors of poor outcome. Pericolic air or no extraluminal air predicted better outcome. Based on these factors, a five-grade staging was created as follows: Stage 1, uncomplicated diverticulitis; Stage 2, complicated diverticulitis with small abscess (<6 cm); Stage 3, complicated diverticulitis with large abscess (≥6 cm) or distant intraperitoneal or retroperitoneal air; Stage 4, Generalized peritonitis without organ dysfunction; Stage 5, generalized peritonitis with organ dysfunction. Mortality was 0, 1%, 3%, 4%, and 32%; need for surgery was 1%, 5%, 46%, 98%, and 100%; and need for intensive care was 0%, 0%, 8%, 11%, and 50%, in Stages 1 to 5, respectively. New staging showed better predictive ability of outcomes compared with earlier classifications in receiver operating characteristic curve analyses. CONCLUSION The proposed staging can be used on all patients both preoperatively and intraoperatively. It takes into account organ dysfunction, which has major influence on survival. The new staging may be easily implemented in daily clinical practice and incorporated in clinical trials. LEVEL OF EVIDENCE Diagnostic study, level II.