Petronella Kettunen

Neuromodulation via Conditional Release of Endocannabinoids in the Spinal Locomotor Network

Endocannabinoids act as retrograde signals to modulate synaptic transmission. Little is known, however, about their significance in integrated network activity underlying motor behavior. We have examined the physiological effects of endocannabinoids in a neuronal network underlying locomotor behavior using the isolated lamprey spinal cord. Our results show that endocannabinoids are released during locomotor activity and participate in setting the baseline burst rate. They are released in response to mGluR1 activation and act as retrograde messengers. This conditional release of endocannabinoids can transform motoneurons and crossing interneurons into modulatory neurons by enabling them to regulate their inhibitory synaptic inputs and thus contribute to the modulation of the locomotor burst frequency. These results provide evidence that endocannabinoid retrograde signaling occurs within the locomotor network and contributes to motor pattern generation and regulation in the spinal cord.

Metabotropic glutamate receptors provide intrinsic modulation of the lamprey locomotor network.

Spinal networks generate the motor pattern underlying locomotion. These are subject to modulatory systems that influence their operation and thereby result in a flexible network organization. In this review, we have summarized the mechanisms by which the different metabotropic glutamate receptor subtypes fine-tune the cellular and synaptic properties and thus underlie intrinsic modulation of the activity of the locomotor network in the lamprey.

Neural circuit activity in freely behaving zebrafish (Danio rerio)

Examining neuronal network activity in freely behaving animals is advantageous for probing the function of the vertebrate central nervous system. Here, we describe a simple, robust technique for monitoring the activity of neural circuits in unfettered, freely behaving zebrafish (Danio rerio). Zebrafish respond to unexpected tactile stimuli with short- or long-latency escape behaviors, which are mediated by distinct neural circuits. Using dipole electrodes immersed in the aquarium, we measured electric field potentials generated in muscle during short- and long-latency escapes. We found that activation of the underlying neural circuits produced unique field potential signatures that are easily recognized and can be repeatedly monitored. In conjunction with behavioral analysis, we used this technique to track changes in the pattern of circuit activation during the first week of development in animals whose trigeminal sensory neurons were unilaterally ablated. One day post-ablation, the frequency of short- and long-latency responses was significantly lower on the ablated side than on the intact side. Three days post-ablation, a significant fraction of escapes evoked by stimuli on the ablated side was improperly executed, with the animal turning towards rather than away from the stimulus. However, the overall response rate remained low. Seven days post-ablation, the frequency of escapes increased dramatically and the percentage of improperly executed escapes declined. Our results demonstrate that trigeminal ablation results in rapid reconfiguration of the escape circuitry, with reinnervation by new sensory neurons and adaptive changes in behavior. This technique is valuable for probing the activity, development, plasticity and regeneration of neural circuits under natural conditions.

Proteomics Profiling of Single Organs from Individual Adult Zebrafish

The model organism zebrafish (Danio rerio) is extensively utilized in studies of developmental biology but is also being investigated in the context of a growing list of human age-related diseases. To facilitate such studies, we here present protein expression patterns of adult zebrafish organs, including blood, brain, fin, heart, intestine, liver, and skeletal muscle. Protein extracts were prepared from the different organs of two zebrafish and analyzed using liquid chromatography coupled to high-resolution tandem mass spectrometry. Zebrafish tissue was digested directly after minimal fractionation and cleaned up (the shotgun approach). Proteins were identified using Mascot software. In total, 1394 proteins were identified of which 644 were nonredundant. Of these, 373 demonstrated an organ-specific expression pattern and 57 had not been shown on protein level before. These data emphasize the need for increased research at the protein level to facilitate the selection of candidate proteins for targeted quantification and to refine systematic genetic network analysis in vertebrate development, biology, and disease.

The zebrafish amyloid precursor protein-b is required for motor neuron guidance and synapse formation

The amyloid precursor protein (APP) is a transmembrane protein mostly recognized for its association with Alzheimers disease. The physiological function of APP is still not completely understood much because of the redundancy between genes in the APP family. In this study we have used zebrafish to study the physiological function of the zebrafish APP homologue, appb, during development. We show that appb is expressed in post-mitotic neurons in the spinal cord. Knockdown of appb by 50-60% results in a behavioral phenotype with increased spontaneous coiling and prolonged touch-induced activity. The spinal cord motor neurons in these embryos show defective formation and axonal outgrowth patterning. Reduction in Appb also results in patterning defects and changed density of pre- and post-synapses in the neuromuscular junctions. Together, our data show that development of functional locomotion in zebrafish depends on a critical role of Appb in the patterning of motor neurons and neuromuscular junctions.

Calcium Imaging in the Zebrafish

The zebrafish (Danio rerio) has emerged as a new model system during the last three decades. The fact that the zebrafish larva is transparent enables sophisticated in vivo imaging. While being the vertebrate, the reduced complexity of its nervous system and small size make it possible to follow large-scale activity in the whole brain. Its genome is sequenced and many genetic and molecular tools have been developed that simplify the study of gene function. Since the mid 1990s, the embryonic development and neuronal function of the larval, and later, adult zebrafish have been studied using calcium imaging methods. The choice of calcium indicator depends on the desired number of cells to study and cell accessibility. Dextran indicators have been used to label cells in the developing embryo from dye injection into the one-cell stage. Dextrans have also been useful for retrograde labeling of spinal cord neurons and cells in the olfactory system. Acetoxymethyl (AM) esters permit labeling of larger areas of tissue such as the tectum, a region responsible for visual processing. Genetically encoded calcium indicators have been expressed in various tissues by the use of cell-specific promoters. These studies have contributed greatly to our understanding of basic biological principles during development and adulthood, and of the function of disease-related genes in a vertebrate system.

Effects of sex and gonadectomy on social investigation and social recognition in mice

BackgroundAn individual’s ability to recognise and pay attention to others is crucial in order to behave appropriately in various social situations. Studies in humans have shown a sex bias in sociability as well as social memory, indicating that females have better face memory and gaze more at the eyes of others, but information about the factors that underpin these differences is sparse. Our aim was therefore to investigate if sociability and social recognition differ between female and male mice, and if so, to what extent gonadal hormones may be involved. Intact and gonadectomised male and female mice were assessed for sociability and social recognition using the three-chambered sociability paradigm, as well as the social discrimination test. Furthermore, we conducted a novel object recognition test, a locomotor activity test and an odour habituation/dishabituation test.ResultsThe present study showed that the ability to recognise other individuals is intact in males with and without gonads, as well as in intact females, whereas it is hampered in gonadectomised females. Additionally, intact male mice displayed more persistent investigatory behaviour compared to the other groups, although the intact females showed elevated basal locomotor activity. In addition, all groups had intact object memory and habituated to odours.ConclusionsOur results suggest that intact male mice investigate conspecifics more than females do, and these differences seem to depend upon circulating hormones released from the testis. As these results seem to contrast what is known from human studies, they should be taken into consideration when using the three-chambered apparatus, and similar paradigms as animal models of social deficits in e.g. autism. Other behavioural tests, and animal models, may be more suitable for translational studies between patients and experimental animals.

Separate signalling mechanisms underlie mGluR1 modulation of leak channels and NMDA receptors in the network underlying locomotion

Metabotropic glutamate receptor subtype 1 (mGluR1) contributes importantly to the activity of the spinal locomotor network. For example, it potentiates NMDA current and inhibits leak conductance in lamprey spinal cord neurons. In this study we examined the signalling pathways underlying the mGluR1 modulation of NMDA receptors and leak channels, respectively. Our results show that mGluR1‐induced potentiation of NMDA current required activation of phospholipase C (PLC) and was independent of the increase in the intracellular Ca2+ concentration because it was unaffected by the Ca2+ chelator BAPTA and by depletion of the internal Ca2+ stores with thapsigargin. We also show that the mGluR1‐mediated inhibition of leak channels is mediated by activation of G‐proteins. Finally, we show that blockade of protein kinase C (PKC) abolished the mGluR1‐induced inhibition of leak current without affecting the potentiation of NMDA receptors. The contribution of mGluR1‐mediated modulation of leak channels to the potentiation of the locomotor cycle frequency was assessed during fictive locomotion. Blockade of PKC significantly decreased the short‐term potentiation of locomotor cycle frequency by mGluR1. These results show that the effects of mGluR1 activation on the two cellular targets, the NMDA receptor and leak channels, are mediated through separate signalling pathways.

Neural Androgen Receptors Modulate Gene Expression and Social Recognition But Not Social Investigation.

The role of sex and androgen receptors (ARs) for social preference and social memory is rather unknown. In this study of mice we compared males, females and males lacking ARs specifically in the nervous system, ARNesDel, with respect to social preference, assessed with the three-chambered apparatus test, and social recognition, assessed with the social discrimination procedure. In the social discrimination test we also evaluated the tentative importance of the sex of the stimulus animal. Novel object recognition and olfaction were investigated to complement the results from the social tests. Gene expression analysis was performed to reveal molecules involved in the effects of sex and androgens on social behaviors. All three test groups showed social preference in the three-chambered apparatus test. In both social tests an AR-independent sexual dimorphism was seen in the persistence of social investigation of female conspecifics, whereas the social interest toward male stimuli mice was similar in all groups. Male and female controls recognized conspecifics independent of their sex, whereas ARNesDel males recognized female but not male stimuli mice. Moreover, the non-social behaviors were not affected by AR deficiency. The gene expression analyses of hypothalamus and amygdala indicated that Oxtr, Cd38, Esr1, Cyp19a1, Ucn3, Crh, and Gtf2i were differentially expressed between the three groups. In conclusion, our results suggest that ARs are required for recognition of male but not female conspecifics, while being dispensable for social investigation toward both sexes. In addition, the AR seems to regulate genes related to oxytocin, estrogen and William’s syndrome.

A Genetic Variant of the Sortilin 1 Gene is Associated with Reduced Risk of Alzheimer's Disease

Alzheimer’s disease (AD) is a neurodegenerative disorder represented by the accumulation of intracellular tau protein and extracellular deposits of amyloid-β (Aβ) in the brain. The gene sortilin 1 (SORT1) has previously been associated with cardiovascular disease in gene association studies. It has also been proposed to be involved in AD pathogenesis through facilitating Aβ clearance by binding apoE/Aβ complexes prior to cellular uptake. However, the neuropathological role of SORT1 in AD is not fully understood. To evaluate the associations between gene variants of SORT1 and risk of AD, we performed genetic analyses in a Swedish case-control cohort. Ten single nucleotide polymorphisms (SNPs), covering the whole SORT1 gene, were selected and genotyped in 620 AD patients and 1107 controls. The SNP rs17646665, located in a non-coding region of the SORT1 gene, remained significantly associated with decreased risk of AD after multiple testing (pc = 0.0061). In addition, other SNPs were found to be nominally associated with risk of AD, as well as altered cognitive function and the CSF biomarker Aβ42, but these associations did not survive correction for multiple testing. The fact that SORT1 has been strongly associated with risk of cardiovascular disease is intriguing as cardiovascular disease is also regarded as a risk factor for AD. Finally, increased knowledge about SORT1 function has a potential to increase our understanding of APOE, the strongest risk factor for AD.