A ventrolateral medulla-midline thalamic circuit for hypoglycemic feeding.

Marked deficits in glucose availability, or glucoprivation, elicit organism-wide counter-regulatory responses whose purpose is to restore glucose homeostasis. However, while catecholamine neurons of the ventrolateral medulla (VLMCA) are thought to orchestrate these responses, the circuit and cellular mechanisms underlying specific counter-regulatory responses are largely unknown. Here, we combined anatomical, imaging, optogenetic and behavioral approaches to interrogate the circuit mechanisms by which VLMCA neurons orchestrate glucoprivation-induced food seeking behavior. Using these approaches, we found that VLMCA neurons form functional connections with nucleus accumbens (NAc)-projecting neurons of the posterior portion of the paraventricular nucleus of the thalamus (pPVT). Importantly, optogenetic manipulations revealed that while activation of VLMCA projections to the pPVT was sufficient to elicit robust feeding behavior in well fed mice, inhibition of VLMCA-pPVT communication significantly impaired glucoprivation-induced feeding while leaving other major counterregulatory responses intact. Collectively our findings identify the VLMCA-pPVT-NAc pathway as a previously-neglected node selectively controlling glucoprivation-induced food seeking. Moreover, by identifying the ventrolateral medulla as a direct source of metabolic information to the midline thalamus, our results support a growing body of literature on the role of the PVT in homeostatic regulation.

Anatomically segregated basal ganglia pathways allow parallel behavioral modulation.

In the basal ganglia (BG), anatomically segregated and topographically organized feedforward circuits are thought to modulate multiple behaviors in parallel. Although topographically arranged BG circuits have been described, the extent to which these relationships are maintained across the BG output nuclei and in downstream targets is unclear. Here, using focal trans-synaptic anterograde tracing, we show that the motor-action-related topographical organization of the striatum is preserved in all BG output nuclei. The topography is also maintained downstream of the BG and in multiple parallel closed loops that provide striatal input. Furthermore, focal activation of two distinct striatal regions induces either licking or turning, consistent with their respective anatomical targets of projection outside of the BG. Our results confirm the parallel model of BG function and suggest that the integration and competition of information relating to different behavior occur largely outside of the BG.

Superior colliculus modulates cortical coding of somatosensory information.

The cortex modulates activity in superior colliculus via a direct projection. What is largely unknown is whether (and if so how) the superior colliculus modulates activity in the cortex. Here, we investigate this issue and show that optogenetic activation of superior colliculus changes the input-output relationship of neurons in somatosensory cortex, enhancing responses to low amplitude whisker deflections. While there is no direct pathway from superior colliculus to somatosensory cortex, we found that activation of superior colliculus drives spiking in the posterior medial (POm) nucleus of the thalamus via a powerful monosynaptic pathway. Furthermore, POm neurons receiving input from superior colliculus provide monosynaptic excitatory input to somatosensory cortex. Silencing POm abolished the capacity of superior colliculus to modulate cortical whisker responses. Our findings indicate that the superior colliculus, which plays a key role in attention, modulates sensory processing in somatosensory cortex via a powerful di-synaptic pathway through the thalamus.

Splice variants of DOMINO control Drosophila circadian behavior and pacemaker neuron maintenance.

Circadian clocks control daily rhythms in behavior and physiology. In Drosophila, the small ventral lateral neurons (sLNvs) expressing PIGMENT DISPERSING FACTOR (PDF) are the master pacemaker neurons generating locomotor rhythms. Despite the importance of sLNvs and PDF in circadian behavior, little is known about factors that control sLNvs maintenance and PDF accumulation. Here, we identify the Drosophila SWI2/SNF2 protein DOMINO (DOM) as a key regulator of circadian behavior. Depletion of DOM in circadian neurons eliminates morning anticipatory activity under light dark cycle and impairs behavioral rhythmicity in constant darkness. Interestingly, the two major splice variants of DOM, DOM-A and DOM-B have distinct circadian functions. DOM-A depletion mainly leads to arrhythmic behavior, while DOM-B knockdown lengthens circadian period without affecting the circadian rhythmicity. Both DOM-A and DOM-B bind to the promoter regions of key pacemaker genes period and timeless, and regulate their protein expression. However, we identify that only DOM-A is required for the maintenance of sLNvs and transcription of pdf. Lastly, constitutive activation of PDF-receptor signaling rescued the arrhythmia and period lengthening of DOM downregulation. Taken together, our findings reveal that two splice variants of DOM play distinct roles in circadian rhythms through regulating abundance of pacemaker proteins and sLNvs maintenance.

Recruitment of GABAergic Interneurons in the Barrel Cortex during Active Tactile Behavior.

Neural computation involves diverse types of GABAergic inhibitory interneurons that are integrated with excitatory (E) neurons into precisely structured circuits. To understand how each neuron type shapes sensory representations, we measured firing patterns of defined types of neurons in the barrel cortex while mice performed an active, whisker-dependent object localization task. Touch excited fast-spiking (FS) interneurons at short latency, followed by activation of E neurons and somatostatin-expressing (SST) interneurons. Touch only weakly modulated vasoactive intestinal polypeptide-expressing (VIP) interneurons. Voluntary whisker movement activated FS neurons in the ventral posteromedial nucleus (VPM) target layers, a subset of SST neurons and a majority of VIP neurons. Together, FS neurons track thalamic input, mediating feedforward inhibition. SST neurons monitor local excitation, providing feedback inhibition. VIP neurons are activated by non-sensory inputs, disinhibiting E and FS neurons. Our data reveal rules of recruitment for interneuron types during behavior, providing foundations for understanding computation in cortical microcircuits.

Feed-forward information and zero-lag synchronization in the sensory thalamocortical circuit are modulated during stimulus perception.

The direction of functional information flow in the sensory thalamocortical circuit may play a role in stimulus perception, but, surprisingly, this process is poorly understood. We addressed this problem by evaluating a directional information measure between simultaneously recorded neurons from somatosensory thalamus (ventral posterolateral nucleus, VPL) and somatosensory cortex (S1) sharing the same cutaneous receptive field while monkeys judged the presence or absence of a tactile stimulus. During stimulus presence, feed-forward information (VPL → S1) increased as a function of the stimulus amplitude, while pure feed-back information (S1 → VPL) was unaffected. In parallel, zero-lag interaction emerged with increasing stimulus amplitude, reflecting externally driven thalamocortical synchronization during stimulus processing. Furthermore, VPL → S1 information decreased during error trials. Also, VPL → S1 and zero-lag interaction decreased when monkeys were not required to report the stimulus presence. These findings provide evidence that both the direction of information flow and the instant synchronization in the sensory thalamocortical circuit play a role in stimulus perception.

Experience-dependent structural plasticity targets dynamic filopodia in regulating dendrite maturation and synaptogenesis.

Highly motile dendritic protrusions are hallmarks of developing neurons. These exploratory filopodia sample the environment and initiate contacts with potential synaptic partners. To understand the role for dynamic filopodia in dendrite morphogenesis and experience-dependent structural plasticity, we analyzed dendrite dynamics, synapse formation, and dendrite volume expansion in developing ventral lateral neurons (LNvs) of the Drosophila larval visual circuit. Our findings reveal the temporal coordination between heightened dendrite dynamics with synaptogenesis in LNvs and illustrate the strong influence imposed by sensory experience on the prevalence of dendritic filopodia, which regulate the formation of synapses and the expansion of dendritic arbors. Using genetic analyses, we further identified Amphiphysin (Amph), a BAR (Bin/Amphiphysin/Rvs) domain-containing protein as a required component for tuning the dynamic state of LNv dendrites and promoting dendrite maturation. Taken together, our study establishes dynamic filopodia as the key cellular target for experience-dependent regulation of dendrite development.

Regulation of feeding by somatostatin neurons in the tuberal nucleus.

The tuberal nucleus (TN) is a surprisingly understudied brain region. We found that somatostatin (SST) neurons in the TN, which is known to exhibit pathological or cytological changes in human neurodegenerative diseases, play a crucial role in regulating feeding in mice. GABAergic tuberal SST (TNSST) neurons were activated by hunger and by the hunger hormone, ghrelin. Activation of TNSST neurons promoted feeding, whereas inhibition reduced it via projections to the paraventricular nucleus and bed nucleus of the stria terminalis. Ablation of TNSST neurons reduced body weight gain and food intake. These findings reveal a previously unknown mechanism of feeding regulation that operates through orexigenic TNSST neurons, providing a new perspective for understanding appetite changes.

Regulation of cortical activity and arousal by the matrix cells of the ventromedial thalamic nucleus.

The "non-specific" ventromedial thalamic nucleus (VM) has long been considered a candidate for mediating cortical arousal due to its diffuse, superficial projections, but direct evidence was lacking. Here, we show in mice that the activity of VM calbindin1-positive matrix cells is high in wake and REM sleep and low in NREM sleep, and increases before cortical activity at the sleep-to-wake transition. Optogenetic stimulation of VM cells rapidly awoke all mice from NREM sleep and consistently caused EEG activation during slow wave anesthesia, while arousal did not occur from REM sleep. Conversely, chemogenetic inhibition of VM decreased wake duration. Optogenetic activation of the "specific" ventral posteromedial nucleus (VPM) did not cause arousal from either NREM or REM sleep. Thus, matrix cells in VM produce arousal and broad cortical activation during NREM sleep and slow wave anesthesia in a way that accounts for the effects classically attributed to "non-specific" thalamic nuclei.

Does layer 4 in the barrel cortex function as a balanced circuit when responding to whisker movements?

Neurons in one barrel in layer 4 (L4) in the mouse vibrissa somatosensory cortex are innervated mostly by neurons from the VPM nucleus and by other neurons within the same barrel. During quiet wakefulness or whisking in air, thalamic inputs vary slowly in time, and excitatory neurons rarely fire. A barrel in L4 contains a modest amount of neurons; the synaptic conductances are not very strong and connections are not sparse. Are the dynamical properties of the L4 circuit similar to those expected from fluctuation-dominated, balanced networks observed for large, strongly coupled and sparse cortical circuits? To resolve this question, we analyze a network of 150 inhibitory parvalbumin-expressing fast-spiking inhibitory interneurons innervated by the VPM thalamus with random connectivity, without or with 1600 low-firing excitatory neurons. Above threshold, the population-average firing rate of inhibitory cortical neurons increases linearly with the thalamic firing rate. The coefficient of variation CV is somewhat less than 1. Moderate levels of synchrony are induced by converging VPM inputs and by inhibitory interaction among neurons. The strengths of excitatory and inhibitory currents during whisking are about three times larger than threshold. We identify values of numbers of presynaptic neurons, synaptic delays between inhibitory neurons, and electrical coupling within the experimentally plausible ranges for which spike synchrony levels are low. Heterogeneity in in-degrees increases the width of the firing rate distribution to the experimentally observed value. We conclude that an L4 circuit in the low-synchrony regime exhibits qualitative dynamical properties similar to those of balanced networks.

Barreloid Borders and Neuronal Activity Shape Panglial Gap Junction-Coupled Networks in the Mouse Thalamus.

The ventral posterior nucleus of the thalamus plays an important role in somatosensory information processing. It contains elongated cellular domains called barreloids, which are the structural basis for the somatotopic organization of vibrissae representation. So far, the organization of glial networks in these barreloid structures and its modulation by neuronal activity has not been studied. We have developed a method to visualize thalamic barreloid fields in acute slices. Combining electrophysiology, immunohistochemistry, and electroporation in transgenic mice with cell type-specific fluorescence labeling, we provide the first structure-function analyses of barreloidal glial gap junction networks. We observed coupled networks, which comprised both astrocytes and oligodendrocytes. The spread of tracers or a fluorescent glucose derivative through these networks was dependent on neuronal activity and limited by the barreloid borders, which were formed by uncoupled or weakly coupled oligodendrocytes. Neuronal somata were distributed homogeneously across barreloid fields with their processes running in parallel to the barreloid borders. Many astrocytes and oligodendrocytes were not part of the panglial networks. Thus, oligodendrocytes are the cellular elements limiting the communicating panglial network to a single barreloid, which might be important to ensure proper metabolic support to active neurons located within a particular vibrissae signaling pathway.

Axons of Individual Dorsal Horn Neurons Bifurcated to Project in Both the Anterolateral and the Postsynaptic Dorsal Column Systems.

Sensory information stimulates receptors of somatosensory system neurons generating a signal that codifies the characteristics of peripheral stimulation. This information reaches the spinal cord and is relayed to supra-spinal structures through two main systems: the postsynaptic dorsal column-medial lemniscal (DC-ML) and the anterolateral (AL) systems. From the classical point of view, the DC-ML has an ipsilateral ascending pathway to the Gracilis (GRA) or Cuneate (CUN) nuclei and the AL has a contralateral ascending pathway to the ventral posterolateral (VPL) thalamic nucleus. These two systems have been the subject of multiple studies that established their independence and interactions. To analyze the ascending projections of L1-L5 spinal dorsal horn neurons in the rat, two retrograde neuronal tracers were injected into the GRA and the VPL. Additionally, an electrophysiological study was performed by applying electrical stimulation at the GRA or VPL and recording antidromic evoked activity in single unit spinal cord cells. Importantly, a subset of spinal dorsal horn neurons exhibited double staining, indicating that these neurons projected to both the GRA and the VPL. These double-stained neurons were located on both sides of the dorsal horn of the spinal cord. The spinal dorsal horn neurons exhibited antidromic and collision activities in response to both GRA and VPL electrical activation. These results show spinal cord neurons with bifurcated bilateral projections to both the DC-ML and AL systems. Based on these results, we named these neurons bilateral and bifurcated cells.

Alpha2delta-1 in SF1+ Neurons of the Ventromedial Hypothalamus Is an Essential Regulator of Glucose and Lipid Homeostasis.

The central mechanisms controlling glucose and lipid homeostasis are inadequately understood. We show that α2δ-1 is an essential regulator of glucose and lipid balance, acting in steroidogenic factor-1 (SF1) neurons of the ventromedial hypothalamus (VMH). These effects are body weight independent and involve regulation of SF1+ neuronal activity and sympathetic output to metabolic tissues. Accordingly, mice with α2δ-1 deletion in SF1 neurons exhibit glucose intolerance, altered lipolysis, and decreased cholesterol content in adipose tissue despite normal energy balance regulation. Profound reductions in the firing rate of SF1 neurons, decreased sympathetic output, and elevated circulating levels of serotonin are associated with these alterations. Normal calcium currents but reduced excitatory postsynaptic currents in mutant SF1 neurons implicate α2δ-1 in the promotion of excitatory synaptogenesis separate from its canonical role as a calcium channel subunit. Collectively, these findings identify an essential mechanism that regulates VMH neuronal activity and glycemic and lipid control and may be a target for tackling metabolic disease.

Dorsal and ventral parts of thalamic nucleus submedius project to different areas of rat orbitofrontal cortex: A single neuron-tracing study using virus vectors.

The rodent orbitofrontal cortex is involved in a variety of cognitive and behavioral functions that require thalamic input to be successfully expressed. Although the thalamic nucleus submedius (Sm) is a major source of afferents to the orbitofrontal cortex, thalamocortical projection from the Sm has not been fully elucidated. In the present study, we first divided the rat Sm into dorsal and ventral parts according to the distribution of vesicular glutamate transporter 2-immunoreactive varicosities, which were somatosensory afferents from the brain stem. Subsequently we investigated dendritic and axonal arborizations of individual dorsal and ventral Sm neurons by visualizing the processes with Sindbis virus vectors expressing membrane-targeted fluorescent proteins. The number of dendritic processes of ventral Sm neurons was greater than that of dorsal Sm neurons. In the cerebral cortex, all the reconstructed Sm neurons sent axons primarily to layers 2-5. Interestingly, dorsal Sm neurons formed a single axon arbor exclusively within the ventrolateral orbital area, whereas ventral Sm neurons made two axon arbors in the lateral orbital and ventral orbital areas simultaneously. The spread of each axon arbor was 500-1000 µm in diameter in the direction tangential to the cortical surface. These results indicate that the dorsal and ventral Sm comprise two distinct thalamocortical pathways. The dorsal Sm pathway relay somatosensory information to the ventrolateral orbital area and may be involved in emotional and aversive aspects of nociceptive information processing, whereas the ventral Sm pathway seems to co-activate distant orbitofrontal cortical areas, and may link their functions under certain circumstances.

Sensory processing of deep tissue nociception in the rat spinal cord and thalamic ventrobasal complex.

Sensory processing of deep somatic tissue constitutes an important component of the nociceptive system, yet associated central processing pathways remain poorly understood. Here, we provide a novel electrophysiological characterization and immunohistochemical analysis of neural activation in the lateral spinal nucleus (LSN). These neurons show evoked activity to deep, but not cutaneous, stimulation. The evoked responses of neurons in the LSN can be sensitized to somatosensory stimulation following intramuscular hypertonic saline, an acute model of muscle pain, suggesting this is an important spinal relay site for the processing of deep tissue nociceptive inputs. Neurons of the thalamic ventrobasal complex (VBC) mediate both cutaneous and deep tissue sensory processing, but in contrast to the lateral spinal nucleus our electrophysiological studies do not suggest the existence of a subgroup of cells that selectively process deep tissue inputs. The sensitization of polymodal and thermospecific VBC neurons to mechanical somatosensory stimulation following acute muscle stimulation with hypertonic saline suggests differential roles of thalamic subpopulations in mediating cutaneous and deep tissue nociception in pathological states. Overall, our studies at both the spinal (lateral spinal nucleus) and supraspinal (thalamic ventrobasal complex) levels suggest a convergence of cutaneous and deep somatosensory inputs onto spinothalamic pathways, which are unmasked by activation of muscle nociceptive afferents to produce consequent phenotypic alterations in spinal and thalamic neural coding of somatosensory stimulation. A better understanding of the sensory pathways involved in deep tissue nociception, as well as the degree of labeled line and convergent pathways for cutaneous and deep somatosensory inputs, is fundamental to developing targeted analgesic therapies for deep pain syndromes.

Synaptic inputs from stroke-injured brain to grafted human stem cell-derived neurons activated by sensory stimuli.

Transplanted neurons derived from stem cells have been proposed to improve function in animal models of human disease by various mechanisms such as neuronal replacement. However, whether the grafted neurons receive functional synaptic inputs from the recipient's brain and integrate into host neural circuitry is unknown. Here we studied the synaptic inputs from the host brain to grafted cortical neurons derived from human induced pluripotent stem cells after transplantation into stroke-injured rat cerebral cortex. Using the rabies virus-based trans-synaptic tracing method and immunoelectron microscopy, we demonstrate that the grafted neurons receive direct synaptic inputs from neurons in different host brain areas located in a pattern similar to that of neurons projecting to the corresponding endogenous cortical neurons in the intact brain. Electrophysiological in vivo recordings from the cortical implants show that physiological sensory stimuli, i.e. cutaneous stimulation of nose and paw, can activate or inhibit spontaneous activity in grafted neurons, indicating that at least some of the afferent inputs are functional. In agreement, we find using patch-clamp recordings that a portion of grafted neurons respond to photostimulation of virally transfected, channelrhodopsin-2-expressing thalamo-cortical axons in acute brain slices. The present study demonstrates, for the first time, that the host brain regulates the activity of grafted neurons, providing strong evidence that transplanted human induced pluripotent stem cell-derived cortical neurons can become incorporated into injured cortical circuitry. Our findings support the idea that these neurons could contribute to functional recovery in stroke and other conditions causing neuronal loss in cerebral cortex.

Mixed-mode oscillations and population bursting in the pre-Bötzinger complex.

This study focuses on computational and theoretical investigations of neuronal activity arising in the pre-Bötzinger complex (pre-BötC), a medullary region generating the inspiratory phase of breathing in mammals. A progressive increase of neuronal excitability in medullary slices containing the pre-BötC produces mixed-mode oscillations (MMOs) characterized by large amplitude population bursts alternating with a series of small amplitude bursts. Using two different computational models, we demonstrate that MMOs emerge within a heterogeneous excitatory neural network because of progressive neuronal recruitment and synchronization. The MMO pattern depends on the distributed neuronal excitability, the density and weights of network interconnections, and the cellular properties underlying endogenous bursting. Critically, the latter should provide a reduction of spiking frequency within neuronal bursts with increasing burst frequency and a dependence of the after-burst recovery period on burst amplitude. Our study highlights a novel mechanism by which heterogeneity naturally leads to complex dynamics in rhythmic neuronal populations.

Inhibitory synaptic transmission from the substantia nigra pars reticulata to the ventral medial thalamus in mice.

The cortico-basal ganglia-thalamic loop circuit is involved in variety of motor, association and limbic functions. The basal ganglia receive neural information from various areas of the cerebral cortex and transfer them back to the frontal and motor cortex via the ventral medial (VM), and the anterior-ventral lateral thalamic complex. The projection from the basal ganglia to the thalamus is GABAergic, and, therefore, the output from the basal ganglia cannot directly evoke excitation in the thalamic nuclei. The mechanism underlying the information transfer via the inhibitory projection remains unclear. To address this issue, we recorded electrophysiological properties of nigro-thalamic synapses from the VM neuron. We developed a nigro-thalamic slice preparation, in which the projection from the substantia nigra pars reticulata (SNr) to VM nucleus is stored, to enable the selective activation of the projection from the SNr. We characterized synaptic properties and membrane properties of the VM neuron, and developed a VM neuron model to simulate the impacts of SNr inputs on VM neuron activity. Neural simulation suggested that the inhibitory projection from SNr can control neural activity in two ways: a disinhibition from the spontaneous nigral inhibition and a ß-band synchronization evoked by combination of excitation and inhibition of SNr activity.

Cerebellar Premotor Output Neurons Collateralize to Innervate the Cerebellar Cortex.

Motor commands computed by the cerebellum are hypothesized to use corollary discharge, or copies of outgoing commands, to accelerate motor corrections. Identifying sources of corollary discharge, therefore, is critical for testing this hypothesis. Here we verified that the pathway from the cerebellar nuclei to the cerebellar cortex in mice includes collaterals of cerebellar premotor output neurons, mapped this collateral pathway, and identified its postsynaptic targets. Following bidirectional tracer injections into a distal target of the cerebellar nuclei, the ventrolateral thalamus, we observed retrogradely labeled somata in the cerebellar nuclei and mossy fiber terminals in the cerebellar granule layer, consistent with collateral branching. Corroborating these observations, bidirectional tracer injections into the cerebellar cortex retrogradely labeled somata in the cerebellar nuclei and boutons in the ventrolateral thalamus. To test whether nuclear output neurons projecting to the red nucleus also collateralize to the cerebellar cortex, we used a Cre-dependent viral approach, avoiding potential confounds of direct red nucleus-to-cerebellum projections. Injections of a Cre-dependent GFP-expressing virus into Ntsr1-Cre mice, which express Cre selectively in the cerebellar nuclei, retrogradely labeled somata in the interposed nucleus, and putative collateral branches terminating as mossy fibers in the cerebellar cortex. Postsynaptic targets of all labeled mossy fiber terminals were identified using immunohistochemical Golgi cell markers and electron microscopic profiles of granule cells, indicating that the collaterals of nuclear output neurons contact both Golgi and granule cells. These results clarify the organization of a subset of nucleocortical projections that constitute an experimentally accessible corollary discharge pathway within the cerebellum.

Anatomically structured burst spiking of thalamic reticular nucleus cells: implications for distinct modulations of sensory processing in lemniscal and non-lemniscal thalamocortical loop circuitries.

The thalamic reticular nucleus (TRN) occupies a highly strategic position to modulate sensory processing in the thalamocortical loop circuitries. It has been shown that TRN visual cells projecting to first- and higher-order thalamic nuclei have distinct levels of burst spiking, suggesting the possibility that the TRN exerts differential influences on information processing in first- and higher-order thalamic nuclei that compose the lemniscal and non-lemniscal sensory systems, respectively. To determine whether this possibility could extend across sensory modalities, the present study examined activities of TRN auditory cells projecting to the ventral and dorsal divisions (first- and higher-order auditory thalamic nuclei) of the medial geniculate nucleus (TRN-MGV and TRN-MGD cells) in anesthetized rats, using juxta-cellular recording and labeling techniques. Burst spiking of TRN-MGV cells consisted of larger numbers of spikes with shorter inter-spike intervals as compared with that of TRN-MGD cells in auditory response evoked by noise burst stimuli. Similar distinctions, although not statistically significant, were observed in spontaneous activity. Furthermore, the features of burst spiking varied in association with the topographies of cell body and terminal field locations. These features of burst spiking are similar to those observed in the two types of TRN visual cells. First- and higher-order thalamic nuclei are known to have distinct levels of burst spiking across sensory modalities. Taken together, it is suggested that the distinctions in burst spiking in the TRN, in conjunction with those in thalamic nuclei, could constitute distinct circuitries for lemniscal and non-lemniscal sensory processing in the thalamocortical loop.