A complete biomechanical model of Hydra contractile behaviors, from neural drive to muscle to movement.

How does neural activity drive muscles to produce behavior? The recent development of genetic lines in Hydra that allow complete calcium imaging of both neuronal and muscle activity, as well as systematic machine learning quantification of behaviors, makes this small cnidarian an ideal model system to understand and model the complete transformation from neural firing to body movements. To achieve this, we have built a neuromechanical model of Hydra's fluid-filled hydrostatic skeleton, showing how drive by neuronal activity activates distinct patterns of muscle activity and body column biomechanics. Our model is based on experimental measurements of neuronal and muscle activity and assumes gap junctional coupling among muscle cells and calcium-dependent force generation by muscles. With these assumptions, we can robustly reproduce a basic set of Hydra's behaviors. We can further explain puzzling experimental observations, including the dual timescale kinetics observed in muscle activation and the engagement of ectodermal and endodermal muscles in different behaviors. This work delineates the spatiotemporal control space of Hydra movement and can serve as a template for future efforts to systematically decipher the transformations in the neural basis of behavior.

Regulation of Spontaneous Propagating Waves in the Embryonic Mouse Brainstem.

Spontaneous activity (SA) modulates many aspects of neural development, including neuronal phenotype, axon path-finding and synaptic connectivity. In the embryonic mouse brainstem, SA initially is recorded in isolated cells at embryonic day (E) 9.5, and 48 h later takes the form of propagating waves. The majority of these waves originate from one midline initiation zone (InZ), which is situated within the developing serotonergic raphe. InZ cells express a t-type calcium channel, are depolarized, and have high membrane resistance, the combination of which allows spontaneous depolarization. Propagating events require signaling at metabotropic 5-HT receptors; a possible source could be 5-HT released by newly differentiating 5-HT neurons. At E11.5, waves propagate throughout the hindbrain, with some events crossing into the midbrain. At E12.5, lateral cells (further than 150 µm from the midline) up-regulate expression of a K channel that increases resting conductance and hyperpolarizes them, preventing the propagation of waves laterally. At the same stage, cells in the isthmus up-regulate t-type calcium channels, permitting more events to cross into the midbrain, some of which form recurring loops of activity that are able to keep intracellular calcium levels high for many minutes. At E13.5, caudal hindbrain cells hyperpolarize utilizing the same K conductance, and 24 h later, at E14.5, the InZ hyperpolarizes and no longer undergoes spontaneous events. Thus, 5-HT receptor-dependent propagating waves in the embryonic brainstem are generated and propagated by regulation of membrane conductance. We discuss these mechanisms, and the possible role of this SA in neuronal development.

Looping circuit: a novel mechanism for prolonged spontaneous [Ca2+]i increases in developing embryonic mouse brainstem.

Most cells maintain [Ca(2+)]i at extremely low levels; calcium entry usually occurs briefly, and within seconds it is cleared. However, at embryonic day 12.5 in the mouse brainstem, trains of spontaneous events occur with [Ca(2+)]i staying close to peak value, well above baseline, for minutes; we termed this 'bash bursts'. Here, we investigate the mechanism of this unusual activity using calcium imaging and electrophysiology. Bash bursts are triggered by an event originating at the mid-line of the rostral hindbrain and are usually the result of that event propagating repeatedly along a defined circular path. The looping circuit can either encompass both the midbrain and hindbrain or remain in the hindbrain only, and the type of loop determines the duration of a single lap time, 5 or 3 s, respectively. Bash bursts are supported by high membrane excitability of mid-line cells and are regulated by persistent inward 'window current' at rest, contributing to spontaneous activity. This looping circuit is an effective means for increasing [Ca(2+)]i at brief, regular intervals. Bash bursts disappear by embryonic day 13.5 via alteration of the looping circuit, curtailing the short epoch of bash bursts. The resulting sustained [Ca(2+)]i may influence development of raphe serotonergic and ventral tegmental dopaminergic neurons by modulating gene expression.

Hyperpolarization of resting membrane potential causes retraction of spontaneous Ca(i)²âº transients during mouse embryonic circuit development.

Abstract Spontaneous activity supports developmental processes in many brain regions during embryogenesis, and the spatial extent and frequency of the spontaneous activity are tightly regulated by stage. In the developing mouse hindbrain, spontaneous activity propagates widely and the waves can cover the entire hindbrain at E11.5. The activity then retracts to waves that are spatially restricted to the rostral midline at E13.5, before disappearing altogether by E15.5. However, the mechanism of retraction is unknown. We studied passive membrane properties of cells that are spatiotemporally relevant to the pattern of retraction in mouse embryonic hindbrain using whole-cell patch clamp and imaging techniques. We find that membrane excitability progressively decreases due to hyperpolarization of resting membrane potential and increased resting conductance density between E11.5 and E15.5, in a spatiotemporal pattern correlated with the retraction sequence. Retraction can be acutely reversed by membrane depolarization at E15.5, and the induced events propagate similarly to spontaneous activity at earlier stages, though without involving gap junctional coupling. Manipulation of [K(+)](o) or [Cl(-)](o) reveals that membrane potential follows E(K) more closely than E(Cl), suggesting a dominant role for K(+) conductance in the membrane hyperpolarization. Reducing membrane excitability by hyperpolarization of the resting membrane potential and increasing resting conductance are effective mechanisms to desynchronize spontaneous activity in a spatiotemporal manner, while allowing information processing to occur at the synaptic and cellular level.

Na(V)1.1 channels are critical for intercellular communication in the suprachiasmatic nucleus and for normal circadian rhythms.

Na(V)1.1 is the primary voltage-gated Na(+) channel in several classes of GABAergic interneurons, and its reduced activity leads to reduced excitability and decreased GABAergic tone. Here, we show that Na(V)1.1 channels are expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus. Mice carrying a heterozygous loss of function mutation in the Scn1a gene (Scn1a(+/-)), which encodes the pore-forming α-subunit of the Na(V)1.1 channel, have longer circadian period than WT mice and lack light-induced phase shifts. In contrast, Scn1a(+/-) mice have exaggerated light-induced negative-masking behavior and normal electroretinogram, suggesting an intact retina light response. Scn1a(+/-) mice show normal light induction of c-Fos and mPer1 mRNA in ventral SCN but impaired gene expression responses in dorsal SCN. Electrical stimulation of the optic chiasm elicits reduced calcium transients and impaired ventro-dorsal communication in SCN neurons from Scn1a(+/-) mice, and this communication is barely detectable in the homozygous gene KO (Scn1a(-/-)). Enhancement of GABAergic transmission with tiagabine plus clonazepam partially rescues the effects of deletion of Na(V)1.1 on circadian period and phase shifting. Our report demonstrates that a specific voltage-gated Na(+) channel and its associated impairment of SCN interneuronal communication lead to major deficits in the function of the master circadian pacemaker. Heterozygous loss of Na(V)1.1 channels is the underlying cause for severe myoclonic epilepsy of infancy; the circadian deficits that we report may contribute to sleep disorders in severe myoclonic epilepsy of infancy patients.

Timing and mechanism of a window of spontaneous activity in embryonic mouse hindbrain development.

Spontaneous activity (SA) in the developing vertebrate brain is required for correct wiring of circuits and networks. In almost every brain region studied to date, SA is recorded during a period of synaptogenesis, and may deploy ionic mechanism(s) that are not expressed in the adult structure. Eventually the conditions in the immature neurons that allow SA are replaced with ion channels found in the mature neuron; this replacement may itself require SA. In the embryonic (E) 11.5 mouse hindbrain, SA is initiated by a subgroup of serotonergic neurons derived from former rhombomeres 2 and 3; SA events propagate rostrally and caudally along the midline, and into the lateral hindbrain. In this review, I describe the properties of mouse hindbrain SA and the developmental window during which it is expressed, summarize the known mechanisms by which SA arises, and describe other brain regions where this SA is similar (chick hindbrain) or influential (mouse midbrain).

Differential expression of membrane conductances underlies spontaneous event initiation by rostral midline neurons in the embryonic mouse hindbrain.

Spontaneous activity is expressed in many developing CNS structures and is crucial in correct network development. Previous work using [Ca(2+)](i) imaging showed that in the embryonic mouse hindbrain spontaneous activity is initiated by a driver population, the serotonergic neurons of the nascent raphe. Serotonergic neurons derived from former rhombomere 2 drive 90% of all hindbrain events at E11.5. We now demonstrate that the electrical correlate of individual events is a spontaneous depolarization, which originates at the rostral midline and drives events laterally. Midline events have both a rapid spike and a large plateau component, while events in lateral tissue comprise only a smaller amplitude plateau. Lateral cells have a large resting conductance and are highly coupled via neurobiotin-permeant gap junctions, while midline cells are significantly less gap junction-coupled and uniquely express a T-type Ca(2+) channel. We propose that the combination of low resting conductance and expression of T-type Ca(2+) current is permissive for midline neurons to acquire the initiator or driver phenotype, while cells without these features cannot drive activity. This demonstrates that expression of specific conductances contributes to the ability to drive spontaneous activity in a developing network.

Spontaneous activity in the developing mouse midbrain driven by an external pacemaker.

Central nervous system (CNS) development depends upon spontaneous activity (SA) to establish networks. We have discovered that the mouse midbrain has SA expressed most robustly at embryonic day (E) 12.5. SA propagation in the midbrain originates in midline serotonergic cell bodies contained within the adjacent hindbrain and then passes through the isthmus along ventral midline serotonergic axons. Once within the midbrain, the wave bifurcates laterally along the isthmic border and then propagates rostrally. Along this trajectory, it is carried by a combination of GABAergic and cholinergic neurons. Removing the hindbrain eliminates SA in the midbrain. Thus, SA in the embryonic midbrain arises from a single identified pacemaker in a separate brain structure, which drives SA waves across both regions of the developing CNS. The midbrain can self-initiate activity upon removal of the hindbrain, but only with pharmacological manipulations that increase excitability. Under these conditions, new initiation foci within the midbrain become active. Anatomical analysis of the development of the serotonergic axons that carry SA from the hindbrain to the midbrain indicates that their increasing elongation during development may control the onset of SA in the midbrain.

Properties and mechanisms of spontaneous activity in the embryonic chick hindbrain.

Spontaneous activity regulates many aspects of central nervous system development. We demonstrate that in the embryonic chick hindbrain, spontaneous activity is expressed between embryonic days (E) 6-9. Over this period the frequency of activity decreases significantly, although the events maintain a consistent rhythm on the timescale of minutes. At E6, the activity is pharmacologically dependent on serotonin, nACh, GABA(A), and glycine input, but not on muscarinic, glutamatergic, or GABA(B) receptor activation. It also depends on gap junctions, t-type calcium channels and TTX-sensitive ion channels. In intact spinal cord-hindbrain preparations, E6 spontaneous events originate in the spinal cord and propagate into lateral hindbrain tissue; midline activity follows the appearance of lateral activity. However, the spinal cord is not required for hindbrain activity. There are two invariant points of origin of activity along the midline, both within the caudal group of serotonin-expressing cell bodies; one point is caudal to the nV exit point while the other is caudal to the nVII exit point. Additional caudal midline points of origin are seen in a minority of cases. Using immunohistochemistry, we show robust differentiation of the serotonergic raphe near the midline at E6, and extensive fiber tracts expressing GAD65/67 and the nAChR in lateral areas; this suggests that the medial activity is dependent on serotonergic neuron activation, while lateral activity requires other transmitters. Although there are differences between species, this activity is highly conserved between mouse and chick, suggesting that developmental event(s) within the hindbrain are dependent on expression of this spontaneous activity.

Ion channel development, spontaneous activity, and activity-dependent development in nerve and muscle cells.

At specific stages of development, nerve and muscle cells generate spontaneous electrical activity that is required for normal maturation of intrinsic excitability and synaptic connectivity. The patterns of this spontaneous activity are not simply immature versions of the mature activity, but rather are highly specialized to initiate and control many aspects of neuronal development. The configuration of voltage- and ligand-gated ion channels that are expressed early in development regulate the timing and waveform of this activity. They also regulate Ca2+ influx during spontaneous activity, which is the first step in triggering activity-dependent developmental programs. For these reasons, the properties of voltage- and ligand-gated ion channels expressed by developing neurons and muscle cells often differ markedly from those of adult cells. When viewed from this perspective, the reasons for complex patterns of ion channel emergence and regression during development become much clearer.

Midline serotonergic neurones contribute to widespread synchronized activity in embryonic mouse hindbrain.

Spontaneous, synchronous activity occurs in motor neurones of the embryonic mouse hindbrain at the stage when rhombomeric segmentation disappears (embryonic day 11.5). The mechanisms generating and synchronizing the activity, however, and the extent to which it is widespread in the hindbrain, are unknown. We show here that spontaneous activity is initiated in the midline of the hindbrain, and propagates laterally to encompass virtually the entire hindbrain synchronously and bilaterally. Separation of the midline region from lateral regions abolishes or slows activity laterally, but not medially. The early differentiating neurones of the midline raphe system are present in the rostral midline and express serotonin at E11.5. Their axons ramify extensively in the marginal zone, cross the midline, and extend at the midline both rostrally into the midbrain and caudally towards the caudal hindbrain. Blockers of serotonin receptors, specifically the 5-HT(2A) receptor, abolish synchronous activity in the hindbrain, while blockers of other neurotransmitter systems, including GABA and glutamate, do not. In addition, the 5-HT(2A) receptor is expressed in the marginal regions in the entire medial-to-lateral extent of the hindbrain and in the midline commissural region. Thus, the serotonergic neurones of the developing midline raphe system may play a role in initiating and propagating spontaneous synchronous activity throughout the hindbrain.

Spontaneous, synchronous electrical activity in neonatal mouse cortical neurones.

Spontaneous [Ca2+]i transients were measured in the mouse neocortex from embryonic day 16 (E16) to postnatal day 6 (P6). On the day of birth (P0), cortical neurones generated widespread, highly synchronous [Ca2+]i transients over large areas. On average, 52% of neurones participated in these transients, and in 20% of slices, an average of 80% participated. These transients were blocked by TTX and nifedipine, indicating that they resulted from Ca2+ influx during electrical activity, and occurred at a mean frequency of 0.91 min(-1). The occurrence of this activity was highly centred at P0: at E16 and P2 an average of only 15% and 24% of neurones, respectively, participated in synchronous transients, and they occurred at much lower frequencies at both E16 and P2 than at P0. The overall frequency of [Ca2+]i transients in individual cells did not change between E16 and P2, just the degree of their synchronicity. The onset of this spontaneous, synchronous activity correlated with a large increase in Na+ current density that occurred just before P0, and its cessation with a large decrease in resting resistance that occurred just after P2. This widespread, synchronous activity may serve a variety of functions in the neonatal nervous system.