Two long-standing problems for superresolution (SR) fluorescence microscopy are high illumination intensity and long acquisition time, which significantly hamper its application for live-cell imaging. Reversibly photoswitchable fluorescent proteins (RSFPs) have made it possible to dramatically lower the illumination intensities in saturated depletion-based SR techniques, such as saturated depletion nonlinear structured illumination microscopy (NL-SIM) and reversible saturable optical fluorescence transition microscopy. The characteristics of RSFPs most critical for SR live-cell imaging include, first, the integrated fluorescence signal across each switching cycle, which depends upon the absorption cross-section, effective quantum yield, and characteristic switching time from the fluorescent "on" to "off" state; second, the fluorescence contrast ratio of on/off states; and third, the photostability under excitation and depletion. Up to now, the RSFPs of the Dronpa and rsEGFP (reversibly switchable EGFP) families have been exploited for SR imaging. However, their limited number of switching cycles, relatively low fluorescence signal, and poor contrast ratio under physiological conditions ultimately restrict their utility in time-lapse live-cell imaging and their ability to reach the desired resolution at a reasonable signal-to-noise ratio. Here, we present a truly monomeric RSFP, Skylan-NS, whose properties are optimized for the recently developed patterned activation NL-SIM, which enables low-intensity (∼100 W/cm(2)) live-cell SR imaging at ∼60-nm resolution at subsecond acquisition times for tens of time points over broad field of view.

Hunger and thirst have distinct goals but control similar ingestive behaviors, and little is known about neural processes that are shared between these behavioral states. We identify glutamatergic neurons in the peri-locus coeruleus (periLC neurons) as a polysynaptic convergence node from separate energy-sensitive and hydration-sensitive cell populations. We develop methods for stable hindbrain calcium imaging in free-moving mice, which show that periLC neurons are tuned to ingestive behaviors and respond similarly to food or water consumption. PeriLC neurons are scalably inhibited by palatability and homeostatic need during consumption. Inhibition of periLC neurons is rewarding and increases consumption by enhancing palatability and prolonging ingestion duration. These properties comprise a double-negative feedback relationship that sustains food or water consumption without affecting food- or water-seeking. PeriLC neurons are a hub between hunger and thirst that specifically controls motivation for food and water ingestion, which is a factor that contributes to hedonic overeating and obesity.

During locomotion in vertebrates, reticulospinal neurons in the hindbrain play critical roles in providing descending excitation to the spinal cord locomotor systems. However, despite the fact that many genes that are used to classify the neuronal identities of neurons in the hindbrain have been identified, the molecular identity of the reticulospinal neurons that are critically involved in locomotor drive is not well understood. Chx10-expressing neurons (V2a neurons) are ipsilaterally projecting glutamatergic neurons in the spinal cord and the hindbrain. Many of the V2a neurons in the hindbrain are known to project to the spinal cord in zebrafish, making hindbrain V2a neurons a prime candidate in descending locomotor drive. Results We investigated the roles of hindbrain V2a neurons using optogenetic and electrophysiological approaches. The forced activation of hindbrain V2a neurons using channelrhodopsin efficiently evoked swimming, whereas the forced inactivation of them using Archearhodopsin3 or Halorhodpsin reliably stopped ongoing swimming. Electrophysiological recordings of two populations of hindbrain reticulospinal V2a neurons showed that they were active during swimming. One population of neurons, small V2a neurons in the caudal hindbrain, fired with low rhythmicity, whereas the other population of neurons, large reticulospinal V2a neurons, called MiV1 neurons, fired more rhythmically. Conclusions These results indicated that hindbrain reticulospinal V2a neurons play critical roles in providing excitation to the spinal locomotor circuits during swimming by providing both tonic and phasic inputs to the circuits.

Neural circuits within the frontal cortex support the flexible selection of goal-directed behaviors by integrating input from brain regions associated with sensory, emotional, episodic, and semantic memory functions. From a connectomics perspective, determining how these disparate afferent inputs target their synapses to specific cell types in the frontal cortex may prove crucial in understanding circuit-level information processing. Here, we used monosynaptic retrograde rabies mapping to examine the distribution of afferent neurons targeting four distinct classes of local inhibitory interneurons and four distinct classes of excitatory projection neurons in mouse infralimbic cortex. Interneurons expressing parvalbumin, somatostatin, or vasoactive intestinal peptide received a large proportion of inputs from hippocampal regions, while interneurons expressing neuron-derived neurotrophic factor received a large proportion of inputs from thalamic regions. A more moderate hippocampal-thalamic dichotomy was found among the inputs targeting excitatory neurons that project to the basolateral amygdala, lateral entorhinal cortex, nucleus reuniens of the thalamus, and the periaqueductal gray. Together, these results show a prominent bias among hippocampal and thalamic afferent systems in their targeting to genetically or anatomically defined sets of frontal cortical neurons. Moreover, they suggest the presence of two distinct local microcircuits that control how different inputs govern frontal cortical information processing.

Cholecystokinin-expressing interneurons (CCKIs) are hypothesized to shape pyramidal cell-firing patterns and regulate network oscillations and related network state transitions. To directly probe their role in the CA1 region, we silenced their activity using optogenetic and chemogenetic tools in mice. Opto-tagged CCKIs revealed a heterogeneous population, and their optogenetic silencing triggered wide disinhibitory network changes affecting both pyramidal cells and other interneurons. CCKI silencing enhanced pyramidal cell burst firing and altered the temporal coding of place cells: theta phase precession was disrupted, whereas sequence reactivation was enhanced. Chemogenetic CCKI silencing did not alter the acquisition of spatial reference memories on the Morris water maze but enhanced the recall of contextual fear memories and enabled selective recall when similar environments were tested. This work suggests the key involvement of CCKIs in the control of place-cell temporal coding and the formation of contextual memories.

A model of acute carbon monoxide poisoning combined with spreading depression (SD) induced metabolic stress was used to examine the protective effects of cerebrolysin (CL) on the development of electrophysiological, behavioral and morphological signs of hypoxic damage. Capillary electrodes were implanted into the neocortex and hippocampus of anesthetized rats which were then exposed for 90 min to breathing of 0.8% to 0.5% CO, while 3 to 4 waves of cortical and hippocampal SD were elicited by microinjections of 5% KCl. Duration of SD-provoked depolarization of cerebral cortex and hippocampus was noted. Nine and 18 to 19 days later propagation of SD waves was recorded with the same electrodes and decrease of their amplitude was used as an index of brain damage which was significant in the hippocampus but not in the cortex. CL-treatment (2.5 ml/kg per day) started after CO administration and continued for 14 days significantly improved hippocampal recovery manifested by increased amplitude of SD waves. Behavioral tests performed 10 and 20 days after CO poisoning in the Morris water maze revealed better performance (escape latency 7 s) in the CL-treated than in untreated animals (14 s). Morphological analysis showed marked damage in the hippocampus consonant with electrophysiological and behavioral findings in the same animals. No apparent histological damage was found in rats exposed to CO inhalation alone without the additional SD-provoked depolarization. It is concluded that chronic CL-treatment enhances recovery of hippocampal tissue after hypoxic damage of intermediate severity.

Hippocampal place cells encode the animal's spatial position. However, it is unknown how different long-range sensory systems affect spatial representations. Here we alternated usage of vision and echolocation in Egyptian fruit bats while recording from single neurons in hippocampal areas CA1 and subiculum. Bats flew back and forth along a linear flight track, employing echolocation in darkness or vision in light. Hippocampal representations remapped between vision and echolocation via two kinds of remapping: subiculum neurons turned on or off, while CA1 neurons shifted their place fields. Interneurons also exhibited strong remapping. Finally, hippocampal place fields were sharper under vision than echolocation, matching the superior sensory resolution of vision over echolocation. Simulating several theoretical models of place-cells suggested that combining sensory information and path integration best explains the experimental sharpening data. In summary, here we show sensory-based global remapping in a mammal, suggesting that the hippocampus does not contain an abstract spatial map but rather a 'cognitive atlas', with multiple maps for different sensory modalities.

Phase precession is a well known phenomenon in which a hippocampal place cell will fire action potentials at successively earlier phases (relative to the theta-band oscillations recorded in the local field potential) as an animal moves through the cell’s receptive field (also known as a place field). We present a model in which CA1 pyramidal cell spiking is driven by dual input components arising from CA3 and EC3. The receptive fields of these two input components overlap but are offset in space from each other such that as the animal moves through the model place field, action potentials are driven first by the CA3 input component and then the EC3 input component. As CA3 synaptic input is known to arrive in CA1 at a later theta phase than EC3 input (Mizuseki et al., 2009; Montgomery et al., 2009), CA1 spiking advances in phase as the model transitions from CA3-driven spiking to EC3-driven spiking. Here spike phase is a function of animal location, placing our results in agreement with many experimental observations characterizing CA1 phase precession (O’Keefe and Recce, 1993; Huxter et al., 2003; Geisler et al., 2007). We predict that experimental manipulations that dramatically enhance or disrupt activity in either of these areas should have a significant effect on phase precession observed in CA1.

The origin of the spatial receptive fields of hippocampal place cells has not been established. A hippocampal CA1 pyramidal cell receives thousands of synaptic inputs, mostly from other spatially tuned neurons; however, how the postsynaptic neuron’s cellular properties determine the response to these inputs during behavior is unknown. We discovered that, contrary to expectations from basic models of place cells and neuronal integration, a small, spatially uniform depolarization of the spatially untuned somatic membrane potential of a silent cell leads to the sudden and reversible emergence of a spatially tuned subthreshold response and place-field spiking. Such gating of inputs by postsynaptic neuronal excitability reveals a cellular mechanism for receptive field origin and may be critical for the formation of hippocampal memory representations.

Relating the function of neuronal cell types to information processing and behavior is a central goal of neuroscience. In the hippocampus, pyramidal cells in CA1 and the subiculum process sensory and motor cues to form a cognitive map encoding spatial, contextual, and emotional information, which they transmit throughout the brain. Do these cells constitute a single class or are there multiple cell types with specialized functions? Using unbiased cluster analysis, we show that there are two morphologically and electrophysiologically distinct principal cell types that carry hippocampal output. We show further that these two cell types are inversely modulated by the synergistic action of glutamate and acetylcholine acting on metabotropic receptors that are central to hippocampal function. Combined with prior connectivity studies, our results support a model of hippocampal processing in which the two pyramidal cell types are predominantly segregated into two parallel pathways that process distinct modalities of information.