Behavioral control through the direct, focal silencing of neuronal activity.

The ability to optically stimulate and inhibit neurons has revolutionized neuroscience research. Here, we present a direct, potent, user-friendly chemical approach for optically silencing neurons. We have rendered saxitoxin (STX), a naturally occurring paralytic agent, transiently inert through chemical protection with a previously undisclosed nitrobenzyl-derived photocleavable group. Exposing the caged toxin, STX-bpc, to a brief (5 ms) pulse of light effects rapid release of a potent STX derivative and transient, spatially precise blockade of voltage-gated sodium channels (NaVs). We demonstrate the efficacy of STX-bpc for parametrically manipulating action potentials in mammalian neurons and brain slice. Additionally, we show the effectiveness of this reagent for silencing neural activity by dissecting sensory-evoked swimming in larval zebrafish. Photo-uncaging of STX-bpc is a straightforward method for non-invasive, reversible, spatiotemporally precise neural silencing without the need for genetic access, thus removing barriers for comparative research.

A brainstem circuit for gravity-guided vertical navigation.

The sensation of gravity anchors our perception of the environment and is crucial for navigation. However, the neural circuits that transform gravity into commands for navigation are undefined. We first determined that larval zebrafish (Danio rerio) navigate vertically by maintaining a consistent heading across a series of upward climb or downward dive bouts. Gravity-blind mutant fish swim with more variable heading and excessive veering, leading to inefficient vertical navigation. After targeted photoablation of ascending vestibular neurons and spinal projecting midbrain neurons, but not vestibulospinal neurons, vertical navigation was impaired. These data define a sensorimotor circuit that uses evolutionarily-conserved brainstem architecture to transform gravitational signals into persistent heading for vertical navigation. The work lays a foundation to understand how vestibular inputs allow animals to move efficiently through their environment.

Multisensory navigational strategies of hatchling fish for dispersal.

Animals influence how they disperse in the environment by sensing local cues and adapting how they move. However, controlling dispersal can present a particular challenge early in life when animals tend to be more limited in their capacities to sense and move. To what extent and by what mechanisms can newly hatched fish control how they disperse? Here, we reveal hatchling sensorimotor mechanisms for controlling dispersal by combining swim tracking and precise sensory manipulations of a model species, zebrafish. In controlled laboratory experiments, if we physically constrained hatchlings or blocked sensations of motion through vision and the lateral line, hatchlings responded by elevating their buoyancy and passively moving with faster surface currents. Complementarily, in stagnant water, hatchlings covered more ground using hyperstable swimming, strongly orienting based on graviception. Using experimentally calibrated hydrodynamic simulations, we show that these hatchling behaviors nearly tripled diffusivity and made dispersal robust to local conditions, suggesting this multisensory strategy may provide important advantages for early life in a variable environment.

A primal role for the vestibular sense in the development of coordinated locomotion.

Mature locomotion requires that animal nervous systems coordinate distinct groups of muscles. The pressures that guide the development of coordination are not well understood. To understand how and why coordination might emerge, we measured the kinematics of spontaneous vertical locomotion across early development in zebrafish (Danio rerio) . We found that zebrafish used their pectoral fins and bodies synergistically during upwards swims. As larvae developed, they changed the way they coordinated fin and body movements, allowing them to climb with increasingly stable postures. This fin-body synergy was absent in vestibular mutants, suggesting sensed imbalance promotes coordinated movements. Similarly, synergies were systematically altered following cerebellar lesions, identifying a neural substrate regulating fin-body coordination. Together these findings link the vestibular sense to the maturation of coordinated locomotion. Developing zebrafish improve postural stability by changing fin-body coordination. We therefore propose that the development of coordinated locomotion is regulated by vestibular sensation.

Balance Sense: Response Motifs that Pervade the Brain.

Measuring how the brain encodes and processes an animal's own motion presents major technical challenges. New approaches demonstrate the viability and merit of measuring vestibular responses throughout the entire brain.

Control of Movement Initiation Underlies the Development of Balance.

Balance arises from the interplay of external forces acting on the body and internally generated movements. Many animal bodies are inherently unstable, necessitating corrective locomotion to maintain stability. Understanding how developing animals come to balance remains a challenge. Here we study the interplay among environment, sensation, and action as balance develops in larval zebrafish. We first model the physical forces that challenge underwater balance and experimentally confirm that larvae are subject to constant destabilization. Larvae propel in swim bouts that, we find, tend to stabilize the body. We confirm the relationship between locomotion and balance by changing larval body composition, exacerbating instability and eliciting more frequent swimming. Intriguingly, developing zebrafish come to control the initiation of locomotion, swimming preferentially when unstable, thus restoring preferred postures. To test the sufficiency of locomotor-driven stabilization and the developing control of movement timing, we incorporate both into a generative model of swimming. Simulated larvae recapitulate observed postures and movement timing across early development, but only when locomotor-driven stabilization and control of movement initiation are both utilized. We conclude the ability to move when unstable is the key developmental improvement to balance in larval zebrafish. Our work informs how emerging sensorimotor ability comes to impact how and why animals move when they do.

Morphology and dendritic maturation of developing principal neurons in the rat basolateral amygdala.

The basolateral nucleus of the amygdala (BLA) assigns emotional valence to sensory stimuli, and many amygdala-dependent behaviors undergo marked development during postnatal life. We recently showed principal neurons in the rat BLA undergo dramatic changes to their electrophysiological properties during the first postnatal month, but no study to date has thoroughly characterized changes to morphology or gene expression that may underlie the functional development of this neuronal population. We addressed this knowledge gap with reconstructions of biocytin-filled principal neurons in the rat BLA at postnatal days 7 (P7), 14, 21, 28, and 60. BLA principal neurons underwent a number of morphological changes, including a twofold increase in soma volume from P7 to P21. Dendritic arbors expanded significantly during the first postnatal month and achieved a mature distribution around P28, in terms of total dendritic length and distance from soma. The number of primary dendrites and branch points were consistent with age, but branch points were found farther from the soma in older animals. Dendrites of BLA principal neurons at P7 had few spines, and spine density increased nearly fivefold by P21. Given the concurrent increase in dendritic material, P60 neurons had approximately 17 times as many total spines as P7 neurons. Together, these developmental transitions in BLA principal neuron morphology help explain a number of concomitant electrophysiological changes during a critical period in amygdala development.

Prenatal stress, regardless of concurrent escitalopram treatment, alters behavior and amygdala gene expression of adolescent female rats.

Depression during pregnancy has been linked to in utero stress and is associated with long-lasting symptoms in offspring, including anxiety, helplessness, attentional deficits, and social withdrawal. Depression is diagnosed in 10-20% of expectant mothers, but the impact of antidepressant treatment on offspring development is not well documented, particularly for females. Here, we used a prenatal stress model of maternal depression to test the hypothesis that in utero antidepressant treatment could mitigate the effects of prenatal stress. We also investigated the effects of prenatal stress and antidepressant treatment on gene expression related to GABAergic and serotonergic neurotransmission in the amygdala, which may underlie behavioral effects of prenatal stress. Nulliparous female rats were implanted with osmotic minipumps delivering clinically-relevant concentrations of escitalopram and mated. Pregnant dams were exposed to 12 days of mixed-modality stressors, and offspring were behaviorally assessed in adolescence (postnatal day 28) and adulthood (beyond day 90) to determine the extent of behavioral change. We found that in utero stress exposure, regardless of escitalopram treatment, increased anxiety-like behavior in adolescent females and profoundly influenced amygdala expression of the chloride transporters KCC2 and NKCC1, which regulate GABAergic function. In contrast, prenatal escitalopram exposure alone elevated amygdala expression of 5-HT1A receptors. In adulthood, anxiety-like behavior returned to baseline and gene expression effects in the amygdala abated, whereas deficits emerged in novel object recognition for rats exposed to stress during gestation. These findings suggest prenatal stress causes age-dependent deficits in anxiety-like behavior and amygdala function in female offspring, regardless of antidepressant exposure.

Prenatal Stress Alters the Development of Socioemotional Behavior and Amygdala Neuron Excitability in Rats.

Prenatal stress (PS) is a risk factor for neurodevelopmental disorders with diverse ages of onset and socioemotional symptoms. Some PS-linked disorders involve characteristic social deficits, such as autism spectrum disorders and schizophrenia, but PS also promotes anxiety disorders. We propose the diversity of symptoms following PS arises from perturbations to early brain development. To this end, we characterized the effects of PS on the developmental trajectory of physiology of the amygdala, a late-developing center for socioemotional control. We found that PS dampened socioemotional behavior and reduced amygdala neuron excitability in offspring during infancy (at postnatal days (P)10, 14, 17 and 21), preadolescence (day 28), and adulthood (day 60). PS offspring in infancy produced fewer isolation-induced vocalizations and in adulthood exhibited less anxiety-like behavior and deficits in social interaction. PS neurons had a more hyperpolarized resting membrane potential from infancy to adulthood and produced fewer action potentials. Moreover, adult amygdala neurons from PS animals expressed larger action potential afterhyperpolarizations and H-current relative to controls, further limiting excitability. Our results suggest that PS can suppress socioemotional behavior throughout development and produce age-specific alterations to amygdala physiology.

Thy1-expressing neurons in the basolateral amygdala may mediate fear inhibition.

Research has identified distinct neuronal circuits within the basolateral amygdala (BLA) that differentially mediate fear expression versus inhibition; however, molecular markers of these populations remain unknown. Here we examine whether optogenetic activation of a cellular subpopulation, which may correlate with the physiologically identified extinction neurons in the BLA, would differentially support fear conditioning versus fear inhibition/extinction. We first molecularly characterized Thy1-channelrhodopsin-2 (Thy1-ChR2-EYFP)-expressing neurons as a subpopulation of glutamatergic pyramidal neurons within the BLA. Optogenetic stimulation of these neurons inhibited a subpopulation of medial central amygdala neurons and shunted excitation from the lateral amygdala. Brief activation of these neurons during fear training disrupted later fear memory in male mice. Optogenetic activation during unreinforced stimulus exposure enhanced extinction retention, but had no effect on fear expression, locomotion, or open-field behavior. Together, these data suggest that the Thy1-expressing subpopulation of BLA pyramidal neurons provide an important molecular and pharmacological target for inhibiting fear and enhancing extinction and for furthering our understanding of the molecular mechanisms of fear processing.

Postnatal maturation of GABAergic transmission in the rat basolateral amygdala.

Many psychiatric disorders, including anxiety and autism spectrum disorders, have early ages of onset and high incidence in juveniles. To better treat and prevent these disorders, it is important to first understand normal development of brain circuits that process emotion. Healthy and maladaptive emotional processing involve the basolateral amygdala (BLA), dysfunction of which has been implicated in numerous psychiatric disorders. Normal function of the adult BLA relies on a fine balance of glutamatergic excitation and GABAergic inhibition. Elsewhere in the brain GABAergic transmission changes throughout development, but little is known about the maturation of GABAergic transmission in the BLA. Here we used whole cell patch-clamp recording and single-cell RT-PCR to study GABAergic transmission in rat BLA principal neurons at postnatal day (P)7, P14, P21, P28, and P35. GABAA currents exhibited a significant twofold reduction in rise time and nearly 25% reduction in decay time constant between P7 and P28. This corresponded with a shift in expression of GABAA receptor subunit mRNA from the α2- to the α1-subunit. The reversal potential for GABAA receptors transitioned from depolarizing to hyperpolarizing with age, from around -55 mV at P7 to -70 mV by P21. There was a corresponding shift in expression of opposing chloride pumps that influence the reversal, from NKCC1 to KCC2. Finally, we observed short-term depression of GABAA postsynaptic currents in immature neurons that was significantly and gradually abolished by P28. These findings reveal that in the developing BLA GABAergic transmission is highly dynamic, reaching maturity at the end of the first postnatal month.

Spike-timing precision and neuronal synchrony are enhanced by an interaction between synaptic inhibition and membrane oscillations in the amygdala.

The basolateral complex of the amygdala (BLA) is a critical component of the neural circuit regulating fear learning. During fear learning and recall, the amygdala and other brain regions, including the hippocampus and prefrontal cortex, exhibit phase-locked oscillations in the high delta/low theta frequency band (∼2-6 Hz) that have been shown to contribute to the learning process. Network oscillations are commonly generated by inhibitory synaptic input that coordinates action potentials in groups of neurons. In the rat BLA, principal neurons spontaneously receive synchronized, inhibitory input in the form of compound, rhythmic, inhibitory postsynaptic potentials (IPSPs), likely originating from burst-firing parvalbumin interneurons. Here we investigated the role of compound IPSPs in the rat and rhesus macaque BLA in regulating action potential synchrony and spike-timing precision. Furthermore, because principal neurons exhibit intrinsic oscillatory properties and resonance between 4 and 5 Hz, in the same frequency band observed during fear, we investigated whether compound IPSPs and intrinsic oscillations interact to promote rhythmic activity in the BLA at this frequency. Using whole-cell patch clamp in brain slices, we demonstrate that compound IPSPs, which occur spontaneously and are synchronized across principal neurons in both the rat and primate BLA, significantly improve spike-timing precision in BLA principal neurons for a window of ∼300 ms following each IPSP. We also show that compound IPSPs coordinate the firing of pairs of BLA principal neurons, and significantly improve spike synchrony for a window of ∼130 ms. Compound IPSPs enhance a 5 Hz calcium-dependent membrane potential oscillation (MPO) in these neurons, likely contributing to the improvement in spike-timing precision and synchronization of spiking. Activation of the cAMP-PKA signaling cascade enhanced the MPO, and inhibition of this cascade blocked the MPO. We discuss these results in the context of spike-timing dependent plasticity and modulation by neurotransmitters important for fear learning, such as dopamine.