Excitation creates a distributed pattern of cortical suppression due to varied recurrent input.

Dense local, recurrent connections are a major feature of cortical circuits, yet how they affect neurons' responses has been unclear, with some studies reporting weak recurrent effects, some reporting amplification, and others indicating local suppression. Here, we show that optogenetic input to mouse V1 excitatory neurons generates salt-and-pepper patterns of both excitation and suppression. Responses in individual neurons are not strongly predicted by that neuron's direct input. A balanced-state network model reconciles a set of diverse observations: the observed dynamics, suppressed responses, decoupling of input and output, and long tail of excited responses. The model shows recurrent excitatory-excitatory connections are strong and also variable across neurons. Together, these results demonstrate that excitatory recurrent connections can have major effects on cortical computations by shaping and changing neurons' responses to input.

Single cell optogenetics reveals attenuation-by-suppression in visual cortical neurons.

The relationship between neurons' input and spiking output is central to brain computation. Studies in vitro and in anesthetized animals suggest nonlinearities emerge in cells' input-output (activation) functions as network activity increases, yet how neurons transform inputs in vivo has been unclear. Here, we characterize cortical principal neurons' activation functions in awake mice using two-photon optogenetics and imaging. We find responses to fixed optogenetic input are nearly unchanged as neurons are excited, reflecting a linear response regime above neurons' resting point. In contrast, responses are dramatically attenuated by suppression. This attenuation is a powerful means to filter inputs arriving to suppressed cells, privileging other inputs arriving to excited neurons. These data have two major implications: first, neural activation functions in vivo accord with the activation functions used in recent machine learning systems, and second, neurons' IO functions can enhance sensory processing by attenuating some inputs while leaving others unchanged.

Amplified cortical neural responses as animals learn to use novel activity patterns.

Cerebral cortex supports representations of the world in patterns of neural activity, used by the brain to make decisions and guide behavior. Past work has found diverse, or limited, changes in the primary sensory cortex in response to learning, suggesting that the key computations might occur in downstream regions. Alternatively, sensory cortical changes may be central to learning. We studied cortical learning by using controlled inputs we insert: we trained mice to recognize entirely novel, non-sensory patterns of cortical activity in the primary visual cortex (V1) created by optogenetic stimulation. As animals learned to use these novel patterns, we found that their detection abilities improved by an order of magnitude or more. The behavioral change was accompanied by large increases in V1 neural responses to fixed optogenetic input. Neural response amplification to novel optogenetic inputs had little effect on existing visual sensory responses. A recurrent cortical model shows that this amplification can be achieved by a small mean shift in recurrent network synaptic strength. Amplification would seem to be desirable to improve decision-making in a detection task; therefore, these results suggest that adult recurrent cortical plasticity plays a significant role in improving behavioral performance during learning.

Bicistronic Expression of a High-Performance Calcium Indicator and Opsin for All-Optical Stimulation and Imaging at Cellular Resolution.

State-of-the-art all-optical systems promise unprecedented access to neural activity in vivo, using multiphoton optogenetics to allow simultaneous imaging and control of activity in selected neurons at cellular resolution. However, to achieve wide use of all-optical stimulation and imaging, simple strategies are needed to robustly and stably express opsins and indicators in the same cells. Here, we describe a bicistronic adeno-associated virus (AAV) that expresses both the fast and bright calcium indicator jGCaMP8s, and a soma-targeted (st) and two-photon-activatable opsin, ChrimsonR. With this method, stChrimsonR stimulation with two-photon holography in the visual cortex of mice drives robust spiking in targeted cells, and neural responses to visual sensory stimuli and spontaneous activity are strong and stable. Cells expressing this bicistronic construct show responses to both photostimulation and visual stimulation that are similar to responses measured from cells expressing the same opsin and indicator via separate viruses. This approach is a simple and robust way to prepare neurons in vivo for two-photon holography and imaging.

Ghrelin infusion into the basolateral amygdala suppresses CTA memory formation in rats via the PI3K/Akt/mTOR and PLC/PKC signaling pathways.

Ghrelin is a circulating orexigenic hormone that promotes feeding behavior and regulates metabolism in humans and rodents. We previously reported that local infusion of ghrelin into the basolateral amygdala (BLA) blocked memory acquisition for conditioned taste aversion (CTA) by activating growth hormone secretagogue receptor 1a. In this study, we further explored the underlying mechanism and signaling pathways mediating ghrelin modulation of CTA memory in rats. Pharmacological agents targeting distinct signaling pathways were infused into the BLA during conditioning. We showed that preadministration of the PI3K inhibitor LY294002 abolished the repressive effect of ghrelin on CTA memory. Moreover, LY294002 pretreatment prevented ghrelin from inhibiting Arc and zif268 mRNA expression in the BLA triggered by CTA memory retrieval. Preadministration of rapamycin eliminated the repressive effect of ghrelin, while Gsk3 inhibitors failed to mimic ghrelin's effect. In addition, PLC and PKC inhibitors microinfused in the BLA blocked ghrelin's repression of CTA acquisition. These results demonstrate that ghrelin signaling in the BLA shapes CTA memory via the PI3K/Akt/mTOR and PLC/PKC pathways. We conducted in vivo multichannel recordings from mouse BLA neurons and found that microinjection of ghrelin (20 µM) suppressed intrinsic excitability. By means of whole-cell recordings from rat brain slices, we showed that bath application of ghrelin (200 nM) had no effect on basal synaptic transmission or synaptic plasticity of BLA pyramidal neurons. Together, this study reveals the mechanism underlying ghrelin-induced interference with CTA memory acquisition in rats, i.e., suppression of intrinsic excitability of BLA principal neurons via the PI3K/Akt/mTOR and PLC/PKC pathways.

Acute But Not Chronic Calorie Restriction Defends against Stress-Related Anxiety and Despair in a GHS-R1a-Dependent Manner.

Ghrelin is an important orexigenic brain-gut hormone that regulates feeding, metabolism and glucose homeostasis in human and rodents at multiple levels. Ghrelin functions by binding to its receptor, the growth hormone secretagogue receptor 1a (GHS-R1a), which is widely expressed both inside and outside of the brain. Both acute and chronic calorie restrictions (CRs) were reported to increase endogenous ghrelin levels and lead to beneficial effects on brain functions, including anti-anxiety effects, anti-depressive effects, and memory improvement. However, the causal relationship and underlying mechanisms are not fully understood. Here, we introduced acute or chronic CR to both GHS-R1a KO (Ghsr-/-) mice and WT (Ghsr+/+) littermates, and investigated anxiety- and despair-related behaviors in the elevated plus maze (EPM), open field (OF) and forced swimming (FS) tests. We found that acute and chronic CR produced similar anxiolytic and anti-despair responses in Ghsr+/+ mice but opposite responses in Ghsr-/- mice. In particular, acute CR enhanced while chronic CR reduced anxiety- and despair-like behaviors in Ghsr-/- mice. Acute CR triggered anxiolytic and anti-despair responses in Ghsr+/+ mice. This effect was abolished by a GHS-R1a antagonist, suggesting a GHS-R1a dependent mechanism. Ad-libitum refeeding masked behavioral responses induced by acute CR in both Ghsr-/- and Ghsr+/+ mice. Altogether, our findings indicate that acute and chronic CRs mitigate anxiety- and despair-like behaviors with different physiological mechanisms, with the former being dependent on endogenous ghrelin release and GHS-R1a signaling, while the latter may not be.

GHS-R1a Deficiency Alleviates Depression-Related Behaviors After Chronic Social Defeat Stress.

Ghrelin is an important orexigenic hormone that regulates feeding, metabolism and glucose homeostasis in human and rodents. Ghrelin functions by binding to its receptor, the growth hormone secretagogue receptor 1a (GHS-R1a), which is widely expressed inside and outside of the brain. Recent studies suggested that acyl-ghrelin, the active form of ghrelin, is a persistent biomarker for chronic stress exposure. However, how ghrelin/GHS-R1a signaling contributes to stress responses and mood regulation remains uncertain. In this study, we applied the chronic social defeat stress (CSDS) paradigm to both GHS-R1a knock-out (Ghsr -/-) mice and littermate control (Ghsr +/+) mice, and then measured their depression- and anxiety-related behaviors. We found that Ghsr + / + mice, but not Ghsr -/- mice, displayed apparent anxiety and depression after CSDS, while two groups mice showed identical behaviors at baseline, non-stress state. By screening the central and peripheral responses of Ghsr -/- mice and Ghsr +/+ mice to chronic stress, we found similar elevations of total ghrelin and adrenocorticotropic hormone (ACTH) in the serum of Ghsr -/- mice and Ghsr +/+ mice after CSDS, but decreased interleukin-6 (IL-6) in the serum of defeated Ghsr -/- mice compared to defeated Ghsr +/+ mice. We also found increased concentration of brain derived neurotropic factor (BDNF) in the hippocampus of Ghsr -/- mice compared to Ghsr +/+ mice after CSDS. The basal levels of ghrelin, ACTH, IL-6, and BDNF were not different between Ghsr -/- mice and Ghsr +/+ mice. Our findings thus suggested that the differential expressions of BDNF and IL-6 after CSDS may contribute to less anxiety and less despair observed in GHS-R1a-deficient mice than in WT control mice. Therefore, ghrelin/GHS-R1a signaling may play a pro-anxiety and pro-depression effect in response to chronic stress, while GHS-R1a deficiency may provide resistance to depressive symptoms of CSDS.

Coding odorant concentration through activation timing between the medial and lateral olfactory bulb.

In mammals, each olfactory bulb (OB) contains a pair of mirror-symmetric glomerular maps organized to reflect odorant receptor identity. The functional implication of maintaining these symmetric medial-lateral maps within each OB remains unclear. Here, using in vivo multielectrode recordings to simultaneously detect odorant-induced activity across the entire OB, we reveal a timing difference in the odorant-evoked onset latencies between the medial and lateral halves. Interestingly, the latencies in the medial and lateral OB decreased at different rates as odorant concentration increased, causing the timing difference between them to also diminish. As a result, output neurons in the medial and lateral OB fired with greater synchrony at higher odorant concentrations. Thus, we propose that temporal differences in activity between the medial and lateral OB can dynamically code odorant concentration, which is subsequently decoded in the olfactory cortex through the integration of synchronous action potentials.

Intrabulbar projecting external tufted cells mediate a timing-based mechanism that dynamically gates olfactory bulb output.

In the mammalian olfactory system, intrabulbar projections (IBPs) mediated by a class of external tufted cells (ET cells) specifically link isofunctional odor columns within the same olfactory bulb. To study the function of these ET cells within the glomerular network, we developed a "hemibulb" preparation that maintains IBPs intact enabling the select activation of ET cells associated with specific glomeruli. Using P2-GFP mice, a line in which the P2 glomeruli are labeled with green fluorescent protein, we recorded from P2 mitral cells (MT cells) while selectively stimulating P2 ET cells. Here, we show that ET-cell activity evokes a slow modulatory (SM) potential within MT cells, which is mediated by the glomerular network and consists of both excitatory and inhibitory components. Interestingly, the timing of the SM potential with respect to olfactory nerve (ON) stimulation can produce converse effects on MT-cell output. When ET-cell activity precedes ON stimulation, the MT-cell response is potentiated; however, when ET-cell activity follows ON stimulation, the MT-cell response is inhibited. Thus, intrabulbar projecting ET cells can shape olfactory bulb output through intraglomerular modulation of MT cells.

Prominent roles for odorant receptor coding sequences in allelic exclusion.

Mammalian odorant receptors (ORs) are crucial for establishing the functional organization of the olfactory system, but the mechanisms controlling their expression remain largely unexplained. Here, we utilized a transgenic approach to explore OR gene regulation. We determined that although olfactory sensory neurons (OSNs) are capable of supporting expression of multiple functional ORs, several levels of control ensure that each neuron normally expresses only a single odorant receptor. Surprisingly, this regulation extends beyond endogenous ORs even preventing expression of transgenes consisting of OR-coding sequences driven by synthetic promoters. Thus, part of the intrinsic feedback system must rely on elements present in the OR-coding sequence. Notably, by expressing the same transgenic ORs precociously in immature neurons, we have overcome this suppression and established a generic method to express any OR in approximately 90% of OSNs. These results provide important insights into the hierarchy of OR gene expression and the vital role of the OR-coding sequence in this regulation.

Dendritic excitability and calcium signalling in the mitral cell distal glomerular tuft.

The processing of odour information starts at the level of the olfactory glomerulus, where the mitral cell distal dendritic tuft not only receives olfactory nerve sensory input but also generates dendrodendritic output to form complicated glomerular synaptic circuits. Analysing the membrane properties and calcium signalling mechanisms in these tiny dendritic branches is crucial for understanding how the glomerular tuft transmits and processes olfactory signals. With the use of two-photon Ca2+ imaging in rat olfactory bulb slices, we found that these distal dendritic branches displayed a significantly larger Ca2+ signal than the soma and primary dendrite trunk. A back-propagating action potential was able to trigger a Ca2+ increase throughout the entire glomerular tuft, indicative of the presence of voltage-gated Ca2+ conductances in all branches at different levels of ramification. In response to a train of action potentials evoked at 60 Hz from the soma, the tuft Ca2+ signal increased linearly with the number of action potentials, suggesting that these glomerular branches were able to support repetitive penetration of Na+ action potentials. When a strong olfactory nerve excitatory input was paired with an inhibition from mitral cell basal dendrites, a small spike-like fast prepotential was revealed at both the soma and distal primary dendrite trunk. Corresponding to this fast prepotential was a Ca2+ increase confined locally within the glomerular tuft. In summary, the mitral cell distal dendritic tuft possesses both Na+ and Ca2+ voltage-dependent conductances which can mediate glomerular Ca2+ responsiveness critical for dendrodendritic output and synaptic plasticity.

Dendritic calcium plateau potentials modulate input-output properties of juxtaglomerular cells in the rat olfactory bulb.

Understanding the intrinsic membrane properties of juxtaglomerular (JG) cells is a necessary step toward understanding the neural basis of olfactory signal processing within the glomeruli. We used patch-clamp recordings and two-photon Ca(2+) imaging in rat olfactory bulb slices to analyze a long-lasting plateau potential generated in JG cells and characterize its functional input-output roles in the glomerular network. The plateau potentials were initially generated by dendritic calcium channels. Bath application of Ni(2+) (250 microM to 1 mM) totally blocked the plateau potential. A local puff of Ni(2+) on JG cell dendrites, but not on the soma, blocked the plateau potentials, indicating the critical contribution of dendritic Ca(2+) channels. Imaging studies with two-photon microscopy showed that a dendritic Ca(2+) increase was always correlated with a dendritic but not a somatic plateau potential. The dendritic Ca(2+) conductance contributed to boosting the initial excitatory postsynaptic potentials (EPSPs) to produce the plateau potential that shunted and reduced the amplitudes of the following EPSPs. This enables the JG cells to act as low-pass filters to convert high-frequency inputs to low-frequency outputs. The low frequency (2.6 +/- 0.8 Hz) of rhythmic plateau potentials appeared to be determined by the intrinsic membrane properties of the JG cell. These properties of the plateau potential may enable JG cells to serve as pacemaker neurons in the synchronization and oscillation of the glomerular network.