The zebrafish histamine H3 receptor modulates aggression, neural activity and forebrain functional connectivity.

AIM: Aggression is a behavioural trait characterized by the intention to harm others for offensive or defensive purposes. Neurotransmitters such as serotonin and dopamine are important mediators of aggression. However, the physiological role of the histaminergic system during this behaviour is currently unclear. Here, we aimed to better understand histaminergic signalling during aggression by characterizing the involvement of the histamine H3 receptor (Hrh3). METHODS: We have generated a novel zebrafish Hrh3 null mutant line using CRISPR-Cas9 genome engineering and investigated behavioural changes and alterations to neural activity using whole brain Ca2+ imaging in zebrafish larvae and ribosomal protein S6 (rpS6) immunohistochemistry in adults. RESULTS: We show that genetic inactivation of the histamine H3 receptor (Hrh3) reduces aggression in zebrafish, an effect that can be reproduced by pharmacological inhibition. In addition, hrh3-/- zebrafish show behavioural impairments consistent with heightened anxiety. Larval in vivo whole brain Ca2+ imaging reveals higher neuronal activity in the forebrain of mutants, but lower activity in specific hindbrain areas and changes in measures of functional connectivity between subregions. Adult hrh3-/- zebrafish display brain region-specific neural activity changes in response to aggression of both key regions of the social decision-making network, and the areas containing histaminergic neurons in the zebrafish brain. CONCLUSION: These results highlight the importance of zebrafish Hrh3 signalling for aggression and anxiety and uncover the brain areas involved. Targeting this receptor might be a potential novel therapeutic route for human conditions characterized by heightened aggression.

Pkd2l1 is required for mechanoception in cerebrospinal fluid-contacting neurons and maintenance of spine curvature.

Defects in cerebrospinal fluid (CSF) flow may contribute to idiopathic scoliosis. However, the mechanisms underlying detection of CSF flow in the central canal of the spinal cord are unknown. Here we demonstrate that CSF flows bidirectionally along the antero-posterior axis in the central canal of zebrafish embryos. In the cfap298tm304 mutant, reduction of cilia motility slows transport posteriorly down the central canal and abolishes spontaneous activity of CSF-contacting neurons (CSF-cNs). Loss of the sensory Pkd2l1 channel nearly abolishes CSF-cN calcium activity and single channel opening. Recording from isolated CSF-cNs in vitro, we show that CSF-cNs are mechanosensory and require Pkd2l1 to respond to pressure. Additionally, adult pkd2l1 mutant zebrafish develop an exaggerated spine curvature, reminiscent of kyphosis in humans. These results indicate that CSF-cNs are mechanosensory cells whose Pkd2l1-driven spontaneous activity reflects CSF flow in vivo. Furthermore, Pkd2l1 in CSF-cNs contributes to maintenance of natural curvature of the spine.

A Low-Cost Method of Skin Swabbing for the Collection of DNA Samples from Small Laboratory Fish.

Fin clipping of live fish under anesthesia is widely used to collect samples for DNA extraction. An alternative, potentially less invasive, approach involves obtaining samples by swabbing the skin of nonanesthetized fish. However, this method has yet to be widely adopted for use in laboratory studies in the biological and biomedical sciences. Here, we compare DNA samples from zebrafish Danio rerio and three-spined sticklebacks Gasterosteus aculeatus collected via fin clipping and skin swabbing techniques, and test a range of DNA extraction methods, including commercially available kits and a lower-cost, in-house method. We verify the method for polymerase chain reaction analysis, and examine the potential risk of cross contamination between individual fish that are netted together. We show that swabbing, which may not require the use of anesthesia or analgesics, offers a reliable alternative to fin clipping. Further work is now required to determine the relative effects of fin clipping and swabbing on the stress responses and subsequent health of fish, and hence the potential of swabbing as a refinement to existing DNA sampling procedures.

Early interneuron dysfunction in ALS: insights from a mutant sod1 zebrafish model.

OBJECTIVE: To determine, when, how, and which neurons initiate the onset of pathophysiology in amyotrophic lateral sclerosis (ALS) using a transgenic mutant sod1 zebrafish model and identify neuroprotective drugs. METHODS: Proteinopathies such as ALS involve mutant proteins that misfold and activate the heat shock stress response (HSR). The HSR is indicative of neuronal stress, and we used a fluorescent hsp70-DsRed reporter in our transgenic zebrafish to track neuronal stress and to measure functional changes in neurons and muscle over the course of the disease. RESULTS: We show that mutant sod1 fish first exhibited the HSR in glycinergic interneurons at 24 hours postfertilization (hpf). By 96 hpf, we observed a significant reduction in spontaneous glycinergic currents induced in spinal motor neurons. The loss of inhibition was followed by increased stress in the motor neurons of symptomatic adults and concurrent morphological changes at the neuromuscular junction (NMJ) indicative of denervation. Riluzole, the only approved ALS drug and apomorphine, an NRF2 activator, reduced the observed early neuronal stress response. INTERPRETATION: The earliest event in the pathophysiology of ALS in the mutant sod1 zebrafish model involves neuronal stress in inhibitory interneurons, resulting from mutant Sod1 expression. This is followed by a reduction in inhibitory input to motor neurons. The loss of inhibitory input may contribute to the later development of neuronal stress in motor neurons and concurrent inability to maintain the NMJ. Riluzole, the approved drug for use in ALS, modulates neuronal stress in interneurons, indicating a novel mechanism of riluzole action.

VANGL1 rare variants associated with neural tube defects affect convergent extension in zebrafish.

In humans, rare non-synonymous variants in the planar cell polarity gene VANGL1 are associated with neural tube defects (NTDs). These variants were hypothesized to be pathogenic based mainly on genetic studies in a large cohort of NTD patients. In this study, we validate the potential pathogenic effect of these mutations in vivo by investigating their effect on convergent extension in zebrafish. Knocking down the expression of tri, the ortholog of Vangl2, using an antisense morpholino (MO), as shown previously, led to a defective convergent extension (CE) manifested by a shortened body axis and widened somites. Co-injection of the human VANGL1 with the tri-MO was able to partially rescue the tri-MO induced phenotype in zebrafish. In contrast, co-injection of two human VANGL1 variants, p.Val239Ile and p.Met328Thr, failed to rescue this phenotype. We next carried out overexpression studies where we measured the ability of the human VANGL1 alleles to induce a CE phenotype when injected at high doses in zebrafish embryos. While overexpressing the wild-type allele led to a severely defective CE, overexpression of either p.Val239Ile or p.Met328Thr variant failed to do so. Results from both tri-MO knockdown/rescue results and overexpression assays suggest that these two variants most likely represent "loss-of-function" alleles that affect protein function during embryonic development. Our study demonstrates a high degree of functional conservation of VANGL genes across evolution and provides a model system for studying potential variants identified in human NTDs.

Mutations in VANGL1 associated with neural-tube defects.

Neural-tube defects such as anencephaly and spina bifida constitute a group of common congenital malformations caused by complex genetic and environmental factors. We have identified three mutations in the VANGL1 gene in patients with familial types (V239I and R274Q) and a sporadic type (M328T) of the disease, including a spontaneous mutation (V239I) appearing in a familial setting. In a protein-protein interaction assay V239I abolished interaction of VANGL1 protein with its binding partners, disheveled-1, -2, and -3. These findings implicate VANGL1 as a risk factor in human neural-tube defects.

Glycine receptors regulate interneuron differentiation during spinal network development.

Glycinergic and GABAergic excitatory chloride-mediated signaling is often the first form of activity to emerge in the nascent nervous system and has been proposed to be essential for several aspects of nervous system development. However, few studies have examined the effects of disrupting glycinergic transmission. Here we perturbed glycinergic transmission in vivo from the onset of development in zebrafish and examined its impact on the formation of the locomotor circuitry. Targeted knockdown of the embryonic glycine receptor alpha2-subunit disrupted rhythm-generating networks and reduced the frequency of spontaneous glycinergic and glutamatergic events. Immunohistochemistry revealed a reduction in the number of spinal interneurons without affecting sensory and motor neurons. This effect was accompanied by a concomitant increase in the number of mitotic cells, suggesting that glycine receptors regulate interneuron differentiation during early development. Despite the loss of many interneurons, a subthreshold rhythm-generating circuit was still capable of forming. These data provide evidence that glycine receptors, in addition to their role in neurotransmission, regulate interneuron differentiation during development of this central neural network.

Rhythmic motor activity evoked by NMDA in the spinal zebrafish larva.

We have examined the localization and activity of the neural circuitry that generates swimming behavior in developing zebrafish that were spinalized to isolate the spinal cord from descending brain inputs. We found that addition of the excitatory amino acid agonist N-methyl-d-aspartate (NMDA) to spinalized zebrafish at 3 days in development induced repeating episodes of rhythmic tail beating activity reminiscent of slow swimming behavior. The neural correlate of this activity, monitored by extracellular recording comprised repeating episodes of rhythmic, rostrocaudally progressing peripheral nerve discharges that alternated between the two sides of the body. Motoneuron recordings revealed an activity pattern comprising a slow oscillatory and a fast synaptic component that was consistent with fictive swimming behavior. Pharmacological and voltage-clamp analysis implicated glycine and glutamate in generation of motoneuron activity. Contralateral alternation of motor activity was disrupted with strychnine, indicating a role for glycine in coordinating left-right alternation during NMDA-induced locomotion. At embryonic stages, while rhythmic synaptic activity patterns could still be evoked in motoneurons, they were typically lower in frequency. Kinematic recordings revealed that prior to 3 days in development, NMDA was unable to reliably generate rhythmic tail beating behavior. We conclude that NMDA induces episodes of rhythmic motor activity in spinalized developing zebrafish that can be monitored physiologically in paralyzed preparations. Therefore as for other vertebrates, the zebrafish central pattern generator is intrinsic to the spinal cord and can operate in isolation provided a tonic source of excitation is given.

Mechanisms underlying the noradrenergic modulation of longitudinal coordination during swimming in Xenopus laevis tadpoles.

Noradrenaline (NA) is a potent modulator of locomotion in many vertebrate nervous systems. When Xenopus tadpoles swim, waves of motor neuron activity alternate across the body and propagate along it with a brief rostro-caudal delay (RC-delay) between segments. We have now investigated the mechanisms underlying the reduction of RC-delay s by NA. When recording from motor neurons caudal to the twelfth postotic cleft, the mid-cycle inhibition was weak and sometimes absent, compared to more rostral locations. NA enhanced and even unmasked inhibition in these caudal neurons and enhanced inhibition in rostral neurons, but to a lesser extent. Consequently, the relative increase in the amplitude of the inhibition was greater in caudal neurons, thus reducing the RC-inhibitory gradient. We next investigated whether NA might affect the electrical properties of neurons, such that enhanced inhibition under NA might promote postinhibitory rebound firing. The synaptic inputs during swimming were simulated using a sustained positive current, superimposed upon which were brief negative currents. When these conditions were held constant NA enhanced the probability of rebound firing--indicating a direct effect on membrane properties--in addition to any indirect effect of enhanced inhibition. We propose that NA preferentially enhances weak caudal inhibition, reducing the inhibitory gradient along the cord. This effect on inhibitory synaptic transmission, comprising parallel pre- and postsynaptic components, will preferentially facilitate rebound firing in caudal neurons, advancing their firing relative to more rostral neurons, whilst additionally increasing the networks ability to sustain the longer cycle periods under NA.

Steps during the development of the zebrafish locomotor network.

This review summarizes recent data from our lab concerning the development of motor activities in the developing zebrafish. The zebrafish is a leading model for studies of vertebrate development because one can obtain a large number of transparent, externally and rapidly developing embryos with motor behaviors that are easy to assess (e.g. for mutagenic screens). The emergence of embryonic motility was studied behaviorally and at the cellular level. The embryonic behaviors appear sequentially and include an early, transient period of spontaneous, alternating tail coilings, followed by responses to touch, and swimming. Patch clamp recording in vivo revealed that an electrically coupled network of a subset of spinal neurons generates spontaneous tail coiling, whereas a chemical (glutamatergic and glycinergic) synaptic drive underlies touch responses and swimming and requires input from the hindbrain. Swimming becomes sustained in larvae once serotonergic neuromodulatory effects are integrated. We end with a brief overview of the genetic tools available for the study of the molecular determinants implicated in locomotor network development in the zebrafish. Combining genetic, behavioral and cellular experimental approaches will advance our understanding of the general principles of locomotor network assembly and function.

Development of the locomotor network in zebrafish.

The zebrafish is a leading model for studies of vertebrate development and genetics. Its embryonic motor behaviors are easy to assess (e.g. for mutagenic screens), the embryos develop rapidly (hatching as larvae at 2 days) and are transparent, permitting calcium imaging and patch clamp recording in vivo. We review primarily the recent advances in understanding the cellular basis for the development of motor activities in the developing zebrafish. The motor activities are generated largely in the spinal cord and hindbrain. In the embryo these segmented structures possess a relatively small number of repeating sets of identifiable neurons. Many types of neurons as well as the two types of muscle cells have been classified based on their morphologies. Some of the molecular signals for cellular differentiation have been identified recently and mutations affecting cell development have been isolated. Embryonic motor behaviors appear in sequence and consist of an early period of transient spontaneous coiling contractions, followed by the emergence of twitching responses to touch, and later by the ability to swim. Coiling contractions are generated by an electrically coupled network of a subset of spinal neurons whereas a chemical (glutamatergic and glycinergic) synaptic drive underlies touch responses and swimming. Swimming becomes sustained in larvae once the neuromodulatory serotonergic system develops. These results indicate many similarities between developing zebrafish and other vertebrates in the properties of the synaptic drive underlying locomotion. Therefore, the zebrafish is a useful preparation for gaining new insights into the development of the neural control of vertebrate locomotion. As the types of neurons, transmitters, receptors and channels used in the locomotor network are being defined, this opens the possibility of combining cellular neurophysiology with forward and reverse molecular genetics to understand the principles of locomotor network assembly and function.

AMRP peptides modulate a novel K(+) current in pleural sensory neurons of Aplysia.

Modulation of Aplysia mechanosensory neurons is thought to underlie plasticity of defensive behaviors that are mediated by these neurons. In the past, identification of modulators that act on the sensory neurons and characterization of their actions has been instrumental in providing insight into the functional role of the sensory neurons in the defensive behaviors. Motivated by this precedent and a recent report of the presence of Aplysia Mytilus inhibitory peptide-related (AMRP) neuropeptides in the neuropile and neurons of the pleural ganglia, we sought to determine whether and how pleural sensory neurons respond to the AMRPs. In cultured pleural sensory neurons under voltage clamp, AMRPs elicited a relatively rapidly developing, then partially desensitizing, outward current. The current exhibited outward rectification; in normal 10 mM K(+), it was outward at membrane potentials more positive than -80 mV but disappeared without reversing at more negative potentials. When external K(+) was elevated to 100 mM, the AMRP-elicited current reversed around -25 mV; the shift in reversal potential was as expected for a current carried primarily by K(+). In the high-K(+) solution, the reversed current began to decrease at potentials more negative than -60 mV, creating a region of negative slope resistance in the I-V relationship. The AMRP-elicited K(+) current was blocked by extremely low concentrations of 4-aminopyridine (4-AP; IC(50) = 1.7 x 10(-7) M) but was not very sensitive to TEA. In cell-attached patches, AMRPs applied outside the patch-thus presumably through a diffusible messenger-increased the activity of a K(+) channel that very likely underlies the macroscopic current. The single-channel current exhibited outward rectification, and the open probability of the channel decreased with hyperpolarization; together, these two factors accounted for the outward rectification of the macroscopic current. Submicromolar 4-AP included in the patch pipette blocked the channel by reducing its open probability without altering the single-channel current. Based on the characteristics of the AMRP-modulated K(+) current, we conclude that it is a novel current that has not been previously described in Aplysia mechanosensory neurons. In addition to this current, two other AMRP-elicited currents, a slow, 4-AP-resistant outward current and a Na(+)-dependent inward current, were occasionally observed in the cultured sensory neurons. Responses consistent with all three currents were observed in sensory neurons in situ in intact pleural ganglia.