Comment on 'Accumbens cholinergic interneurons dynamically promote dopamine release and enable motivation'.

Acetylcholine is widely believed to modulate the release of dopamine in the striatum of mammals. Experiments in brain slices clearly show that synchronous activation of striatal cholinergic interneurons is sufficient to drive dopamine release via axo-axonal stimulation of nicotinic acetylcholine receptors. However, evidence for this mechanism in vivo has been less forthcoming. Mohebi, Collins and Berke recently reported that, in awake behaving rats, optogenetic activation of striatal cholinergic interneurons with blue light readily evokes dopamine release measured with the red fluorescent sensor RdLight1 (Mohebi et al., 2023). Here, we show that blue light alone alters the fluorescent properties of RdLight1 in a manner that may be misconstrued as phasic dopamine release, and that this artefactual photoactivation can account for the effects attributed to cholinergic interneurons. Our findings indicate that measurements of dopamine using the red-shifted fluorescent sensor RdLight1 should be interpreted with caution when combined with optogenetics. In light of this and other publications that did not observe large acetylcholine-evoked dopamine transients in vivo, the conditions under which such release occurs in behaving animals remain unknown.

A Cre-dependent reporter mouse for quantitative real-time imaging of protein kinase A activity dynamics.

Intracellular signaling dynamics play a crucial role in cell function. Protein kinase A (PKA) is a key signaling molecule that has diverse functions, from regulating metabolism and brain activity to guiding development and cancer progression. We previously developed an optical reporter, FLIM-AKAR, that allows for quantitative imaging of PKA activity via fluorescence lifetime imaging microscopy and photometry. However, using viral infection or electroporation for the delivery of FLIM-AKAR is invasive and results in variable expression. Here, we developed a reporter mouse, FL-AK, which expresses FLIM-AKAR in a Cre-dependent manner from the ROSA26 locus. FL-AK provides robust and consistent expression of FLIM-AKAR over time. Functionally, the mouse line reports an increase in PKA activity in response to activation of both Gαs and Gαq-coupled receptors in brain slices. In vivo, FL-AK reports PKA phosphorylation in response to neuromodulator receptor activation. Thus, FL-AK provides a quantitative, robust, and flexible method to reveal the dynamics of PKA activity in diverse cell types.

Longevity, demographic characteristics, and socio-economic status are linked to triiodothyronine levels in the general population.

The hypothalamic-pituitary-thyroid (HPT) axis is fundamental to human biology, exerting central control over energy expenditure and body temperature. However, the consequences of normal physiologic HPT-axis variation in populations without diagnosed thyroid disease are poorly understood. Using nationally representative data from the 2007 to 2012 National Health and Nutrition Examination Survey, we explore relationships with demographic characteristics, longevity, and socio-economic factors. We find much larger variation across age in free T3 than other HPT-axis hormones. T3 and T4 have opposite relationships to mortality: free T3 is inversely related and free T4 is positively related to the likelihood of death. Free T3 and household income are negatively related, particularly at lower incomes. Finally, free T3 among older adults is associated with labor both in terms of unemployment and hours worked. Physiologic TSH/T4 explain only 1.7% of T3 variation, and neither are appreciably correlated to socio-economic outcomes. Taken together, our data suggest an unappreciated complexity of the HPT-axis signaling cascade broadly such that TSH and T4 may not be accurate surrogates of free T3. Furthermore, we find that subclinical variation in the HPT-axis effector hormone T3 is an important and overlooked factor linking socio-economic forces, human biology, and aging.

Cortical reactivations predict future sensory responses.

Many theories of offline memory consolidation posit that the pattern of neurons activated during a salient sensory experience will be faithfully reactivated, thereby stabilizing the pattern1,2. However, sensory-evoked patterns are not stable but, instead, drift across repeated experiences3-6. Here, to investigate the relationship between reactivations and the drift of sensory representations, we imaged the calcium activity of thousands of excitatory neurons in the mouse lateral visual cortex. During the minute after a visual stimulus, we observed transient, stimulus-specific reactivations, often coupled with hippocampal sharp-wave ripples. Stimulus-specific reactivations were abolished by local cortical silencing during the preceding stimulus. Reactivations early in a session systematically differed from the pattern evoked by the previous stimulus-they were more similar to future stimulus response patterns, thereby predicting both within-day and across-day representational drift. In particular, neurons that participated proportionally more or less in early stimulus reactivations than in stimulus response patterns gradually increased or decreased their future stimulus responses, respectively. Indeed, we could accurately predict future changes in stimulus responses and the separation of responses to distinct stimuli using only the rate and content of reactivations. Thus, reactivations may contribute to a gradual drift and separation in sensory cortical response patterns, thereby enhancing sensory discrimination7.

A Cre-dependent reporter mouse for quantitative real-time imaging of Protein Kinase A activity dynamics.

Intracellular signaling dynamics play a crucial role in cell function. Protein kinase A (PKA) is a key signaling molecule that has diverse functions, from regulating metabolism and brain activity to guiding development and cancer progression. We previously developed an optical reporter, FLIM-AKAR, that allows for quantitative imaging of PKA activity via fluorescence lifetime imaging microscopy and photometry. However, using viral infection or electroporation for the delivery of FLIM-AKAR is invasive, cannot easily target sparse or hard-to-transfect/infect cell types, and results in variable expression. Here, we developed a reporter mouse, FL-AK, which expresses FLIM-AKAR in a Cre-dependent manner from the ROSA26 locus. FL-AK provides robust and consistent expression of FLIM-AKAR over time. Functionally, the mouse line reports an increase in PKA activity in response to activation of both Gαs and Gαq-coupled receptors in brain slices. In vivo, FL-AK reports PKA phosphorylation in response to neuromodulator receptor activation. Thus, FL-AK provides a quantitative, robust, and flexible method to reveal the dynamics of PKA activity in diverse cell types.

Striatum supports fast learning but not memory recall.

Animals learn to carry out motor actions in specific sensory contexts to achieve goals. The striatum has been implicated in producing sensory-motor associations, yet its contribution to memory formation or recall is not clear. To investigate the contribution of striatum to these processes, mice were taught to associate a cue, consisting of optogenetic activation of striatum-projecting neurons in visual cortex, with forelimb reaches to access food pellets. As necessary to direct learning, striatal neural activity encoded both the sensory context and outcome of reaching. With training, the rate of cued reaching increased, but brief optogenetic inhibition of striatal activity arrested learning and prevented trial-to-trial improvements in performance. However, the same manipulation did not affect performance improvements already consolidated into short- (within an hour) or long-term (across days) memories. Hence, striatal activity is necessary for trial-to-trial improvements in task performance, leading to plasticity in other brain areas that mediate memory recall.

Thyroid hormone rewires cortical circuits to coordinate body-wide metabolism and exploratory drive.

Animals adapt to varying environmental conditions by modifying the function of their internal organs, including the brain. To be adaptive, alterations in behavior must be coordinated with the functional state of organs throughout the body. Here we find that thyroid hormone- a prominent regulator of metabolism in many peripheral organs- activates cell-type specific transcriptional programs in anterior regions of cortex of adult mice via direct activation of thyroid hormone receptors. These programs are enriched for axon-guidance genes in glutamatergic projection neurons, synaptic regulators across both astrocytes and neurons, and pro-myelination factors in oligodendrocytes, suggesting widespread remodeling of cortical circuits. Indeed, whole-cell electrophysiology recordings revealed that thyroid hormone induces local transcriptional programs that rewire cortical neural circuits via pre-synaptic mechanisms, resulting in increased excitatory drive with a concomitant sensitization of recruited inhibition. We find that thyroid hormone bidirectionally regulates innate exploratory behaviors and that the transcriptionally mediated circuit changes in anterior cortex causally promote exploratory decision-making. Thus, thyroid hormone acts directly on adult cerebral cortex to coordinate exploratory behaviors with whole-body metabolic state.

Dopamine and glutamate regulate striatal acetylcholine in decision-making.

Striatal dopamine and acetylcholine are essential for the selection and reinforcement of motor actions and decision-making1. In vitro studies have revealed an intrastriatal circuit in which acetylcholine, released by cholinergic interneurons (CINs), drives the release of dopamine, and dopamine, in turn, inhibits the activity of CINs through dopamine D2 receptors (D2Rs). Whether and how this circuit contributes to striatal function in vivo is largely unknown. Here, to define the role of this circuit in a living system, we monitored acetylcholine and dopamine signals in the ventrolateral striatum of mice performing a reward-based decision-making task. We establish that dopamine and acetylcholine exhibit multiphasic and anticorrelated transients that are modulated by decision history and reward outcome. Dopamine dynamics and reward encoding do not require the release of acetylcholine by CINs. However, dopamine inhibits acetylcholine transients in a D2R-dependent manner, and loss of this regulation impairs decision-making. To determine how other striatal inputs shape acetylcholine signals, we assessed the contribution of cortical and thalamic projections, and found that glutamate release from both sources is required for acetylcholine release. Altogether, we uncover a dynamic relationship between dopamine and acetylcholine during decision-making, and reveal multiple modes of CIN regulation. These findings deepen our understanding of the neurochemical basis of decision-making and behaviour.

Synaptic and circuit functions of multitransmitter neurons in the mammalian brain.

Neurons in the mammalian brain are not limited to releasing a single neurotransmitter but often release multiple neurotransmitters onto postsynaptic cells. Here, we review recent findings of multitransmitter neurons found throughout the mammalian central nervous system. We highlight recent technological innovations that have made the identification of new multitransmitter neurons and the study of their synaptic properties possible. We also focus on mechanisms and molecular constituents required for neurotransmitter corelease at the axon terminal and synaptic vesicle, as well as some possible functions of multitransmitter neurons in diverse brain circuits. We expect that these approaches will lead to new insights into the mechanism and function of multitransmitter neurons, their role in circuits, and their contribution to normal and pathological brain function.

Developmental regulation of GABAergic gene expression in forebrain cholinergic neurons.

Acetylcholine and GABA are often co-released, including from VIP-expressing neurons of the cortex, cortically-projecting neurons of the globus pallidus externus and basal forebrain, and hippocampal-projecting neurons of the medial septum. The co-release of the functionally antagonistic neurotransmitters GABA and acetylcholine (ACh) greatly expands the possible functional effects of cholinergic neurons and provides an additional exogenous source of inhibition to the cortex. Transgene expression suggests that nearly all forebrain cholinergic neurons in mice at some point in development express Slc32a1, which encodes the vesicular GABA transporter (VGAT). To determine the degree of co-expression of GABA and Ach handling proteins, we measured expression in adult mice of Slc32a1, Gad1 and Gad2 (which encode GAD67 and GAD65, respectively, the GABA synthetic enzymes) in cholinergic neurons using fluorescent in situ hybridization. We found that only a subset of cholinergic neurons express the necessary machinery for GABA release at a single time in adult mice. This suggests that GABA co-release from cholinergic neurons is dynamic and potentially developmentally regulated. By measuring expression of Slc32a1, Gad1, Gad2, and Chat in the basal forebrain and medial septum in mice from post-natal day 0 to 28, we noted abundant yet variable expressions of GABAergic markers across early development, which are subsequently downregulated in adulthood. This is in contrast with the forebrain-projecting pedunculopontine nucleus, which showed no evidence of co-expression of GABAergic genes. These results suggest that expression of GABA signaling machinery in the cortically-projecting cholinergic system peaks during early development before settling at a non-zero level that is maintained through adulthood.

Sub-clinical triiodothyronine levels predict health, demographic, and socioeconomic outcomes.

The Hypothalamic-Pituitary-Thyroid (HPT) axis is fundamental to human biology, exerting central control over energy expenditure, metabolic rate, and body temperature. However, the consequences of "normal" physiologic HPT-axis variation in non-clinical populations are poorly understood. Using nationally-representative data from the 2007-2012 NHANES, we explore relationships with demographics, mortality, and socio-economic factors. We find much larger variation across age in free T3 than other HPT-axis hormones. T3 and T4 have opposite effects on mortality: free T3 is inversely related and free T4 is positively related with likelihood of death. Free T3 and household income are negatively related, particularly at lower incomes. Finally, free T3 among older adults is associated with labor both on the extensive margin (unemployment) and intensive margin (hours worked). Physiologic TSH/T4 explain only 1% of T3 variation, and neither are appreciably correlated to socio-economic outcomes. Taken together, our data suggest an unappreciated complexity and non-linearity of the HPT-axis signaling cascade broadly such that TSH and T4 may not be accurate surrogates of free T3. Furthermore, we find that sub-clinical variation in the HPT-axis effector hormone T3 is an important and overlooked factor linking socio-economic forces, human biology, and aging.

A NPAS4-NuA4 complex couples synaptic activity to DNA repair.

Neuronal activity is crucial for adaptive circuit remodelling but poses an inherent risk to the stability of the genome across the long lifespan of postmitotic neurons1-5. Whether neurons have acquired specialized genome protection mechanisms that enable them to withstand decades of potentially damaging stimuli during periods of heightened activity is unknown. Here we identify an activity-dependent DNA repair mechanism in which a new form of the NuA4-TIP60 chromatin modifier assembles in activated neurons around the inducible, neuronal-specific transcription factor NPAS4. We purify this complex from the brain and demonstrate its functions in eliciting activity-dependent changes to neuronal transcriptomes and circuitry. By characterizing the landscape of activity-induced DNA double-strand breaks in the brain, we show that NPAS4-NuA4 binds to recurrently damaged regulatory elements and recruits additional DNA repair machinery to stimulate their repair. Gene regulatory elements bound by NPAS4-NuA4 are partially protected against age-dependent accumulation of somatic mutations. Impaired NPAS4-NuA4 signalling leads to a cascade of cellular defects, including dysregulated activity-dependent transcriptional responses, loss of control over neuronal inhibition and genome instability, which all culminate to reduce organismal lifespan. In addition, mutations in several components of the NuA4 complex are reported to lead to neurodevelopmental and autism spectrum disorders. Together, these findings identify a neuronal-specific complex that couples neuronal activity directly to genome preservation, the disruption of which may contribute to developmental disorders, neurodegeneration and ageing.

Spontaneous behaviour is structured by reinforcement without explicit reward.

Spontaneous animal behaviour is built from action modules that are concatenated by the brain into sequences1,2. However, the neural mechanisms that guide the composition of naturalistic, self-motivated behaviour remain unknown. Here we show that dopamine systematically fluctuates in the dorsolateral striatum (DLS) as mice spontaneously express sub-second behavioural modules, despite the absence of task structure, sensory cues or exogenous reward. Photometric recordings and calibrated closed-loop optogenetic manipulations during open field behaviour demonstrate that DLS dopamine fluctuations increase sequence variation over seconds, reinforce the use of associated behavioural modules over minutes, and modulate the vigour with which modules are expressed, without directly influencing movement initiation or moment-to-moment kinematics. Although the reinforcing effects of optogenetic DLS dopamine manipulations vary across behavioural modules and individual mice, these differences are well predicted by observed variation in the relationships between endogenous dopamine and module use. Consistent with the possibility that DLS dopamine fluctuations act as a teaching signal, mice build sequences during exploration as if to maximize dopamine. Together, these findings suggest a model in which the same circuits and computations that govern action choices in structured tasks have a key role in sculpting the content of unconstrained, high-dimensional, spontaneous behaviour.

Neuronal-Activity Dependent Mechanisms of Small Cell Lung Cancer Progression.

Neural activity is increasingly recognized as a critical regulator of cancer growth. In the brain, neuronal activity robustly influences glioma growth both through paracrine mechanisms and through electrochemical integration of malignant cells into neural circuitry via neuron-to-glioma synapses, while perisynaptic neurotransmitter signaling drives breast cancer brain metastasis growth. Outside of the CNS, innervation of tumors such as prostate, breast, pancreatic and gastrointestinal cancers by peripheral nerves similarly regulates cancer progression. However, the extent to which the nervous system regulates lung cancer progression, either in the lung or when metastatic to brain, is largely unexplored. Small cell lung cancer (SCLC) is a lethal high-grade neuroendocrine tumor that exhibits a strong propensity to metastasize to the brain. Here we demonstrate that, similar to glioma, metastatic SCLC cells in the brain co-opt neuronal activity-regulated mechanisms to stimulate growth and progression. Optogenetic stimulation of cortical neuronal activity drives proliferation and invasion of SCLC brain metastases. In the brain, SCLC cells exhibit electrical currents and consequent calcium transients in response to neuronal activity, and direct SCLC cell membrane depolarization is sufficient to promote the growth of SCLC tumors. In the lung, vagus nerve transection markedly inhibits primary lung tumor formation, progression and metastasis, highlighting a critical role for innervation in overall SCLC initiation and progression. Taken together, these studies illustrate that neuronal activity plays a crucial role in dictating SCLC pathogenesis in both primary and metastatic sites.

Ascertaining cells' synaptic connections and RNA expression simultaneously with barcoded rabies virus libraries.

Brain function depends on synaptic connections between specific neuron types, yet systematic descriptions of synaptic networks and their molecular properties are not readily available. Here, we introduce SBARRO (Synaptic Barcode Analysis by Retrograde Rabies ReadOut), a method that uses single-cell RNA sequencing to reveal directional, monosynaptic relationships based on the paths of a barcoded rabies virus from its "starter" postsynaptic cell to that cell's presynaptic partners. Thousands of these partner relationships can be ascertained in a single experiment, alongside genome-wide RNAs. We use SBARRO to describe synaptic networks formed by diverse mouse brain cell types in vitro, finding that different cell types have presynaptic networks with differences in average size and cell type composition. Patterns of RNA expression suggest that functioning synapses are critical for rabies virus uptake. By tracking individual rabies clones across cells, SBARRO offers new opportunities to map the synaptic organization of neural circuits.

A single-cell atlas of the cycling murine ovary.

The estrous cycle is regulated by rhythmic endocrine interactions of the nervous and reproductive systems, which coordinate the hormonal and ovulatory functions of the ovary. Folliculogenesis and follicle progression require the orchestrated response of a variety of cell types to allow the maturation of the follicle and its sequela, ovulation, corpus luteum formation, and ovulatory wound repair. Little is known about the cell state dynamics of the ovary during the estrous cycle and the paracrine factors that help coordinate this process. Herein, we used single-cell RNA sequencing to evaluate the transcriptome of >34,000 cells of the adult mouse ovary and describe the transcriptional changes that occur across the normal estrous cycle and other reproductive states to build a comprehensive dynamic atlas of murine ovarian cell types and states.

Wireless multi-lateral optofluidic microsystems for real-time programmable optogenetics and photopharmacology.

In vivo optogenetics and photopharmacology are two techniques for controlling neuronal activity that have immense potential in neuroscience research. Their applications in tether-free groups of animals have been limited in part due to tools availability. Here, we present a wireless, battery-free, programable multilateral optofluidic platform with user-selected modalities for optogenetics, pharmacology and photopharmacology. This system features mechanically compliant microfluidic and electronic interconnects, capabilities for dynamic control over the rates of drug delivery and real-time programmability, simultaneously for up to 256 separate devices in a single cage environment. Our behavioral experiments demonstrate control of motor behaviors in grouped mice through in vivo optogenetics with co-located gene delivery and controlled photolysis of caged glutamate. These optofluidic systems may expand the scope of wireless techniques to study neural processing in animal models.

Tapered fibertrodes for optoelectrical neural interfacing in small brain volumes with reduced artefacts.

Deciphering the neural patterns underlying brain functions is essential to understanding how neurons are organized into networks. This deciphering has been greatly facilitated by optogenetics and its combination with optoelectronic devices to control neural activity with millisecond temporal resolution and cell type specificity. However, targeting small brain volumes causes photoelectric artefacts, in particular when light emission and recording sites are close to each other. We take advantage of the photonic properties of tapered fibres to develop integrated 'fibertrodes' able to optically activate small brain volumes with abated photoelectric noise. Electrodes are positioned very close to light emitting points by non-planar microfabrication, with angled light emission allowing the simultaneous optogenetic manipulation and electrical read-out of one to three neurons, with no photoelectric artefacts, in vivo. The unconventional implementation of two-photon polymerization on the curved taper edge enables the fabrication of recoding sites all around the implant, making fibertrodes a promising complement to planar microimplants.

Neurophotonic tools for microscopic measurements and manipulation: status report.

Neurophotonics was launched in 2014 coinciding with the launch of the BRAIN Initiative focused on development of technologies for advancement of neuroscience. For the last seven years, Neurophotonics' agenda has been well aligned with this focus on neurotechnologies featuring new optical methods and tools applicable to brain studies. While the BRAIN Initiative 2.0 is pivoting towards applications of these novel tools in the quest to understand the brain, this status report reviews an extensive and diverse toolkit of novel methods to explore brain function that have emerged from the BRAIN Initiative and related large-scale efforts for measurement and manipulation of brain structure and function. Here, we focus on neurophotonic tools mostly applicable to animal studies. A companion report, scheduled to appear later this year, will cover diffuse optical imaging methods applicable to noninvasive human studies. For each domain, we outline the current state-of-the-art of the respective technologies, identify the areas where innovation is needed, and provide an outlook for the future directions.