Konstantinos Ampatzis

Principles governing recruitment of motoneurons during swimming in zebrafish

Locomotor movements are coordinated by a network of neurons that produces sequential muscle activation. Different motoneurons need to be recruited in an orderly manner to generate movement with appropriate speed and force. However, the mechanisms governing recruitment order have not been fully clarified. Using an in vitro juvenile/adult zebrafish brainstem-spinal cord preparation, we found that motoneurons were organized into four pools with specific topographic locations and were incrementally recruited to produce swimming at different frequencies. The threshold of recruitment was not dictated by the input resistance of motoneurons, but was instead set by a combination of specific biophysical properties and the strength of the synaptic currents. Our results provide insights into the cellular and synaptic computations governing recruitment of motoneurons during locomotion.

Separate Microcircuit Modules of Distinct V2a Interneurons and Motoneurons Control the Speed of Locomotion

Spinal circuits generate locomotion with variable speed as circumstances demand. These circuits have been assumed to convey equal and uniform excitation to all motoneurons whose input resistance dictates their activation sequence. However, the precise connectivity pattern between excitatory premotor circuits and the different motoneuron types has remained unclear. Here, we generate a connectivity map in adult zebrafish between the V2a excitatory interneurons and slow, intermediate, and fast motoneurons. We show that the locomotor network does not consist of a uniform circuit as previously assumed. Instead, it can be deconstructed into three separate microcircuit modules with distinct V2a interneuron subclasses driving slow, intermediate, or fast motoneurons. This modular design enables the increase of locomotor speed by sequentially adding microcircuit layers from slow to intermediate and fast. Thus, this principle of organization of vertebrate spinal circuits represents an intrinsic mechanism to increase the locomotor speed by incrementally engaging different motor units.

Pattern of Innervation and Recruitment of Different Classes of Motoneurons in Adult Zebrafish

In vertebrates, spinal circuits drive rhythmic firing in motoneurons in the appropriate sequence to produce locomotor movements. These circuits become active early during development and mature gradually to acquire the flexibility necessary to accommodate the increased behavioral repertoire of adult animals. The focus here is to elucidate how different pools of motoneurons are organized and recruited and how membrane properties contribute to their mode of operation. For this purpose, we have used the in vitro preparation of adult zebrafish. We show that different motoneuron pools are organized in a somatotopic fashion in the motor column related to the type of muscle fibers (slow, intermediate, fast) they innervate. During swimming, the different motoneuron pools are recruited in a stepwise manner from slow, to intermediate, to fast to cover the full range of locomotor frequencies seen in intact animals. The spike threshold, filtering properties, and firing patterns of the different motoneuron pools are graded in a manner that relates to their order of recruitment. Our results thus show that motoneurons in adult zebrafish are organized into distinct modules, each with defined locations, properties, and recruitment patterns tuned to precisely match the muscle properties and hence produce swimming of different speeds and modalities.

Optogenetic Activation of Excitatory Premotor Interneurons Is Sufficient to Generate Coordinated Locomotor Activity in Larval Zebrafish

Neural networks in the spinal cord can generate locomotion in the absence of rhythmic input from higher brain structures or sensory feedback because they contain an intrinsic source of excitation. However, the molecular identity of the spinal interneurons underlying the excitatory drive within the locomotor circuit has remained unclear. Using optogenetics, we show that activation of a molecularly defined class of ipsilateral premotor interneurons elicits locomotion. These interneurons represent the excitatory module of the locomotor networks and are sufficient to produce a coordinated swimming pattern in zebrafish. They correspond to the V2a interneuron class and express the transcription factor Chx10. They produce sufficient excitatory drive within the spinal networks to generate coordinated locomotor activity. Therefore, our results define the V2a interneurons as the excitatory module within the spinal locomotor networks that is sufficient to initiate and maintain locomotor activity.

Motor neurons control locomotor circuit function retrogradely via gap junctions

Motor neurons are the final stage of neural processing for the execution of motor behaviours. Traditionally, motor neurons have been viewed as the ‘final common pathway’, serving as passive recipients merely conveying to the muscles the final motor program generated by upstream interneuron circuits. Here we reveal an unforeseen role of motor neurons in controlling the locomotor circuit function via gap junctions in zebrafish. These gap junctions mediate a retrograde analogue propagation of voltage fluctuations from motor neurons to control the synaptic release and recruitment of the upstream V2a interneurons that drive locomotion. Selective inhibition of motor neurons during ongoing locomotion de-recruits V2a interneurons and strongly influences locomotor circuit function. Rather than acting as separate units, gap junctions unite motor neurons and V2a interneurons into functional ensembles endowed with a retrograde analogue computation essential for locomotor rhythm generation. These results show that motor neurons are not a passive recipient of motor commands but an integral component of the neural circuits responsible for motor behaviour.

Sex differences in adult cell proliferation within the zebrafish (Danio rerio) cerebellum

It has been reported that neurons generated in the adult brain show sex‐specific differences in several brain regions of lower vertebrates and mammals. The present study questioned whether cell proliferation and survival in the adult zebrafish (Danio rerio) cerebellum, the most mitotically active area of adult teleost brain, is sexually differentiated. Adult zebrafish were treated with the thymidine analogue 5′‐bromo‐2′‐deoxyuridine (BrdU) and allowed to survive for 24 h (short‐term) and for 21 days (long‐term). BrdU immunohistochemistry allowed visualization of cells incorporating BrdU at the S phase of mitosis. At short‐term survival, male zebrafish had a higher number of labelled cells at proliferation sites of the molecular layer of corpus cerebelli (CCe) and the granular layer of the caudal lobe of the cerebellum (LCa) than did females. In long‐term survival, BrdU‐positive cells were found at their final destination, but only the granular layer of the medial division of the valvula cerebelli showed sex‐specific differences in the number of labelled cells. This higher mitotic activity in male cerebellum might be related to sex‐specific motor behaviour observed in male zebrafish. To investigate the role of programmed cell death, the terminal deoxynucleotidyl‐mediated dUTP nick‐end‐labelling (TUNEL) method was applied. The vast majority of apoptotic figures occurred in the granular cell layer of valvula and CCe, only in a few cases within the BrdU‐retaining cells. Apoptosis was found specifically at the sites of the final destination of proliferating cells, indicating that the close relation of cell birth and death might represent a possible plasticity mechanism in the adult zebrafish cerebellum.

Regional distribution and cellular localization of β2‐adrenoceptors in the adult zebrafish brain (Danio rerio)

The β2‐adrenergic receptors (ARs) are G‐protein‐coupled receptors that mediate the physiological responses to adrenaline and noradrenaline. The present study aimed to determine the regional distribution of β2‐ARs in the adult zebrafish (Danio rerio) brain by means of in vitro autoradiographic and immunohistochemical methods. The immunohistochemical localization of β2‐ARs, in agreement with the quantitative β‐adrenoceptor autoradiography, showed a wide distribution of β2‐ARs in the adult zebrafish brain. The cerebellum and the dorsal zone of periventricular hypothalamus exhibited the highest density of [3H]CGP‐12177 binding sites and β2‐AR immunoreactivity. Neuronal cells strongly stained for β2‐ARs were found in the periventricular ventral telencephalic area, magnocellular and parvocellular superficial pretectal nuclei (PSm, PSp), occulomotor nucleus (NIII), locus coeruleus (LC), medial octavolateral nucleus (MON), magnocellular octaval nucleus (MaON) reticular formation (SRF, IMRF, IRF), and ganglionic cell layer of cerebellum. Interestingly, in most cases (NIII, LC, MON, MaON, SRF, IMRF, ganglionic cerebellar layer) β2‐ARs were colocalized with α2A‐ARs in the same neuron, suggesting their interaction for mediating the physiological functions of nor/adrenaline. Moderate to low labeling of β2‐ARs was found in neurons in dorsal telencephalic area, optic tectum (TeO), torus semicircularis (TS), and periventricular gray zone of optic tectum (PGZ). In addition to neuronal, glial expression of β2‐ARs was found in astrocytic fibers located in the central gray and dorsal rhombencephalic midline, in close relation to the ventricle. The autoradiographic and immunohistochemical distribution pattern of β2‐ARs in the adult zebrafish brain further support the conserved profile of adrenergic/noradrenergic system through vertebrate brain evolution. J. Comp. Neurol. 518:1418–1441, 2010.

Neuronal and glial localization of α2A‐adrenoceptors in the adult zebrafish (Danio rerio) brain

The α2A‐adrenoceptor (AR) subtype, a G protein‐coupled receptor located both pre‐ and postsynaptically, mediates adrenaline/noradrenaline functions. The present study aimed to determine the α2A‐AR distribution in the adult zebrafish (Danio rerio) brain by means of immunocytochemistry. Detailed mapping showed labeling of α2A‐ARs, in neuropil, neuronal somata and fibers, glial processes, and blood vessels. A high density of α2A‐AR immunoreactivity was found in the ventral telencephalic area, preoptic, pretectal, hypothalamic areas, torus semicircularis, oculomotor nucleus (NIII), locus coreruleus (LC), medial raphe, medial octavolateralis nucleus (MON), magnocellular octaval nucleus (MaON), reticular formation (SRF, IMRF, IRF), rhombencephalic nerves and roots (DV, V, VII, VIII, X), and cerebellar Purkinje cell layer. Moderate levels of α2A‐ARs were observed in the medial and central zone nuclei of dorsal telencephalic area, in the periventricular gray zone of optic tectum, in the dorsomedial part of optic tectum layers, and in the molecular and granular layers of all cerebellum subdivisions. Glial processes were found to express α2A‐ARs in rhombencephalon, intermingled with neuronal fibers. Medium‐sized neurons were labeled in telencephalic, diencephalic, and mesencephlic areas, whereas densely labeled large neurons were found in rhombencephalon, locus coeruleus, reticular formation, oculomotor area, medial octavolateralis and magnocellular octaval nuclei, and Purkinje cell somata. The functional role of α2A‐ARs on neurons and glial processes is not known at present; however, their strong relation to the ventricular system, somatosensory nuclei, and nerves supports a possible regulatory role of α2A‐ARs in autonomic functions, nerve output, and sensory integration in adult zebrafish brain. J. Comp. Neurol. 508:72–93, 2008.

A Hardwired Circuit Supplemented with Endocannabinoids Encodes Behavioral Choice in Zebrafish

Animals constantly make behavioral choices to facilitate moving efficiently through their environment. When faced with a threat, animals make decisions in the midst of other ongoing behaviors through a context-dependent integration of sensory stimuli. In vertebrates, the mechanisms underlying behavioral selection are poorly understood. Here, we show that ongoing swimming in zebrafish is suppressed by escape. The selection of escape over swimming is mediated by switching between two distinct motoneuron pools. A hardwired circuit mediates this switch by acting as a clutch-like mechanism to disengage the swimming motoneuron pool and engage the escape motoneuron pool. Threshold for escape initiation is lowered and swimming suppression is prolonged by endocannabinoid neuromodulation. Thus, our results reveal a novel cellular mechanism involving a hardwired circuit supplemented with endocannabinoids acting as a clutch-like mechanism to engage/disengage distinct motor pools to ensure behavioral selection and a smooth execution of motor action sequences in a vertebrate system.

Spinal cholinergic interneurons differentially control motoneuron excitability and alter the locomotor network operational range

While cholinergic neuromodulation is important for locomotor circuit operation, the specific neuronal mechanisms that acetylcholine employs to regulate and fine-tune the speed of locomotion are largely unknown. Here, we show that cholinergic interneurons are present in the zebrafish spinal cord and differentially control the excitability of distinct classes of motoneurons (slow, intermediate and fast) in a muscarinic dependent manner. Moreover, we reveal that m2-type muscarinic acetylcholine receptors (mAChRs) are present in fast and intermediate motoneurons, but not in the slow motoneurons, and that their activation decreases neuronal firing. We also reveal a strong correlation between the muscarinic receptor configuration on motoneurons and the ability of the animals to locomote at different speeds, which might serve as a plasticity mechanism to alter the operational range of the locomotor networks. These unexpected findings provide new insights into the functional flexibility of motoneurons and how they execute locomotion at different speeds.