Vasoactive Intestinal Polypeptide Neurons Research Articles

Circadian rhythm mechanism in the suprachiasmatic nucleus and its relation to the olfactory system.

Animals need sleep, and the suprachiasmatic nucleus, the center of the circadian rhythm, plays an important role in determining the timing of sleep. The main input to the suprachiasmatic nucleus is the retinohypothalamic tract, with additional inputs from the intergeniculate leaflet pathway, the serotonergic afferent from the raphe, and other hypothalamic regions. Within the suprachiasmatic nucleus, two of the major subtypes are vasoactive intestinal polypeptide (VIP)-positive neurons and arginine-vasopressin (AVP)-positive neurons. VIP neurons are important for light entrainment and synchronization of suprachiasmatic nucleus neurons, whereas AVP neurons are important for circadian period determination. Output targets of the suprachiasmatic nucleus include the hypothalamus (subparaventricular zone, paraventricular hypothalamic nucleus, preoptic area, and medial hypothalamus), the thalamus (paraventricular thalamic nuclei), and lateral septum. The suprachiasmatic nucleus also sends information through several brain regions to the pineal gland. The olfactory bulb is thought to be able to generate a circadian rhythm without the suprachiasmatic nucleus. Some reports indicate that circadian rhythms of the olfactory bulb and olfactory cortex exist in the absence of the suprachiasmatic nucleus, but another report claims the influence of the suprachiasmatic nucleus. The regulation of circadian rhythms by sensory inputs other than light stimuli, including olfaction, has not been well studied and further progress is expected.

Phenotyping of light-activated neurons in the mouse SCN based on the expression of FOS and EGR1.

Light-sensitive neurons are located in the ventral and central core of the suprachiasmatic nucleus (SCN), whereas stably oscillating clock neurons are found mainly in the dorsal shell. Signals between the SCN core and shell are believed to play an important role in light entrainment. Core neurons express vasoactive intestinal polypeptide (VIP), gastrin-releasing peptide (GRP), and Neuroglobin (Ngb), whereas the shell neurons express vasopressin (AVP), prokineticin 2, and the VIP type 2 (VPAC2) receptor. In rodents, light has a phase-shifting capacity at night, which induces rapid and transient expression of the EGR1 and FOS in the SCN. Methods: The present study used immunohistochemical staining of FOS, EGR1, and phenotypical markers of SCN neurons (VIP, AVP, Ngb) to identify subtypes/populations of light-responsive neurons at early night. Results: Double immunohistochemistry and cell counting were used to evaluate the number of SCN neurons expressing FOS and EGR1 in the SCN. The number of neurons expressing either EGR1 or FOS was higher than the total number of neurons co-storing EGR1 and FOS. Of the total number of light-responsive cells, 42% expressed only EGR1, 43% expressed only FOS, and 15% expressed both EGR1 and FOS. Light-responsive VIP neurons represented only 31% of all VIP neurons, and EGR1 represents the largest group of light-responsive VIP neurons (18%). VIP neurons expressing only FOS represented 1% of the total light-responsive VIP neurons. 81% of the Ngb neurons in the mouse SCN were light-responsive, and of these neurons expressing only EGR1 after light stimulation represented 44%, whereas 24% expressed FOS. Although most light-responsive neurons are found in the core of the SCN, 29% of the AVP neurons in the shell were light-responsive, of which 8% expressed EGR1, 10% expressed FOS, and 11% co-expressed both EGR1 and FOS after light stimulation. Discussion: Our analysis revealed cell-specific differences in light responsiveness between different peptidergic and Ngb-expressing neurons in different compartments of the mouse SCN, indicating that light activates diverse neuronal networks in the SCN, some of which participate in photoentrainment.

Cholecystokinin neurons in mouse suprachiasmatic nucleus regulate the robustness of circadian clock

The suprachiasmatic nucleus (SCN) can generate robust circadian behaviors in mammals under different environments, but the underlying neural mechanisms remained unclear. Here, we showed that the activities of cholecystokinin (CCK) neurons in the mouse SCN preceded the onset of behavioral activities under different photoperiods. CCK-neuron-deficient mice displayed shortened free-running periods, failed to compress their activities under a long photoperiod, and developed rapid splitting or became arrhythmic under constant light. Furthermore, unlike vasoactive intestinal polypeptide (VIP) neurons, CCK neurons are not directly light sensitive, but their activation can elicit phase advance and counter light-induced phase delay mediated by VIP neurons. Under long photoperiods, the impact of CCK neurons on SCN dominates over that of VIP neurons. Finally, we found that the slow-responding CCK neurons control the rate of recovery during jet lag. Together, our results demonstrated that SCN CCK neurons are crucial for the robustness and plasticity of the mammalian circadian clock.

The local and long-range input landscape of inhibitory neurons in mouse auditory cortex.

Roughly 20% of the neurons in the mouse cortex are inhibitory interneurons (INs). Of these, the three major subtypes are parvalbumin (PV), somatostatin (SST), and vasoactive intestinal polypeptide (VIP) expressing neurons. We used monosynaptic rabies tracing to compare the presynaptic input landscape onto these three IN subtypes in the mouse primary auditory cortex (A1). We compared both local patterns of monosynaptic inputs as well as long-range input patterns. The local monosynaptic input landscape to SST neurons was more widespread as compared to PV and VIP neurons. The brain-wide input landscape was rich and heterogeneous with >40 brain regions connecting to all the three INs subtypes from both hemispheres. The general pattern of the long-range input landscape was similar among the groups of INs. Nevertheless, a few differences could be identified. At low resolution, the proportion of local versus long-range inputs was smaller for PV neurons. At mesoscale resolution, we found fewer inputs from temporal association area to VIP INs, and more inputs to SST neurons from basal forebrain and lateral amygdala. Our work can be used as a resource for a quantitative comparison of the location and level of inputs impinging onto discrete populations of neurons in mouse A1.

Cortex-wide response mode of VIP-expressing inhibitory neurons by reward and punishment.

Neocortex is classically divided into distinct areas, each specializing in different function, but all could benefit from reinforcement feedback to inform and update local processing. Yet it remains elusive how global signals like reward and punishment are represented in local cortical computations. Previously, we identified a cortical neuron type, vasoactive intestinal polypeptide (VIP)-expressing interneurons, in auditory cortex that is recruited by behavioral reinforcers and mediates disinhibitory control by inhibiting other inhibitory neurons. As the same disinhibitory cortical circuit is present virtually throughout cortex, we wondered whether VIP neurons are likewise recruited by reinforcers throughout cortex. We monitored VIP neural activity in dozens of cortical regions using three-dimensional random access two-photon microscopy and fiber photometry while mice learned an auditory discrimination task. We found that reward and punishment during initial learning produce rapid, cortex-wide activation of most VIP interneurons. This global recruitment mode showed variations in temporal dynamics in individual neurons and across areas. Neither the weak sensory tuning of VIP interneurons in visual cortex nor their arousal state modulation was fully predictive of reinforcer responses. We suggest that the global response mode of cortical VIP interneurons supports a cell-type-specific circuit mechanism by which organism-level information about reinforcers regulates local circuit processing and plasticity.

Neurotransmitter phenotype and axonal projection patterns of VIP-expressing neurons in the inferior colliculus

Neurons in the inferior colliculus (IC), the midbrain hub of the central auditory pathway, send ascending and descending projections to other auditory brain regions, as well as projections to other sensory and non-sensory brain regions. However, the axonal projection patterns of individual classes of IC neurons remain largely unknown. Vasoactive intestinal polypeptide (VIP) is a neuropeptide expressed by subsets of neurons in many brain regions. We recently identified a class of IC stellate neurons that we called VIP neurons because they are labeled by tdTomato (tdT) expression in VIP-IRES-Cre x Ai14 mice. Here, using fluorescence in situ hybridization, we found that tdT+ neurons in VIP-IRES-Cre x Ai14 mice express Vglut2, a marker of glutamatergic neurons, and VIP, suggesting that VIP neurons use both glutamatergic and VIPergic signaling to influence their postsynaptic targets. Next, using viral transfections with a Cre-dependent eGFP construct, we labeled the axonal projections of VIP neurons. As a group, VIP neurons project intrinsically, within the ipsilateral and contralateral IC, and extrinsically to all the major targets of the IC. Within the auditory system, VIP neurons sent axons and formed axonal boutons in higher centers, including the medial geniculate nucleus and the nucleus of the brachium of the IC. Less dense projections terminated in lower centers, including the nuclei of the lateral lemniscus, superior olivary complex, and dorsal cochlear nucleus. VIP neurons also project to several non-auditory brain regions, including the superior colliculus, periaqueductal gray, and cuneiform nucleus. The diversity of VIP projections compared to the homogeneity of VIP neuron intrinsic properties suggests that VIP neurons play a conserved role at the microcircuit level, likely involving neuromodulation through glutamatergic and VIPergic signaling, but support diverse functions at the systems level through their participation in different projection pathways.

Vigilance and Behavioral State-Dependent Modulation of Cortical Neuronal Activity throughout the Sleep/Wake Cycle.

GABAergic inhibitory neurons, through their molecular, anatomic, and physiological diversity, provide a substrate for the modulation of ongoing cortical circuit activity throughout the sleep/wake cycle. Here, we investigated neuronal activity dynamics of parvalbumin (PV), vasoactive intestinal polypeptide (VIP), and somatostatin (SST) neurons in naturally sleeping head-restrained mice at the level of layer 2/3 of the primary somatosensory barrel cortex of mice. Through calcium imaging and targeted single-unit loose-patch or whole-cell recordings, we found that PV action potential firing activity was largest during both rapid eye movement (REM) and nonrapid eye movement (NREM) sleep stages, that VIP neurons were most active during REM sleep, and that the overall activity of SST neurons remained stable throughout the sleep/wake cycle. Analysis of neuronal activity dynamics uncovered rapid decreases in PV cell firing at wake onset followed by a progressive recovery during wake. Simultaneous local field potential (LFP) recordings further revealed that except for SST neurons, a large proportion of neurons were modulated by ongoing delta and theta oscillations. During NREM sleep spindles, PV and SST activity increased and decreased, respectively. Finally, we uncovered the presence of whisking behavior in mice during REM sleep and show that the activity of VIP and SST is differentially modulated during awake and sleeping whisking bouts, which may provide a neuronal substrate for internal brain representations occurring during sleep.SIGNIFICANCE STATEMENT In the sensory cortex, the balance between excitation and inhibition is believed to be highly dynamic throughout the sleep/wake cycle, shaping the response of cortical circuits to external stimuli while allowing the formation of newly encoded memory. Using in vivo two-photon calcium imaging or targeted single-unit recordings combined with LFP recordings, we describe the vigilance state and whisking-behavior-dependent activity of excitatory pyramidal and inhibitory GABAergic neurons in the supragranular layers of mouse somatosensory cortex. Interneuronal activity was found to be differentially modulated by ongoing delta and theta waves, sleep spindles, and a novel type of whisking observed during REM sleep, potentially providing a neuronal substrate for internal brain representations occurring during sleep.

Plastic embedding for precise imaging of large-scale biological tissues labeled with multiple fluorescent dyes and proteins.

Resin embedding of multi-color labeled whole organs is the primary step to preserve structural information for visualization of fine structures in three dimensions. It is essential to study the morphological characteristics, spatial and positional relationships of the millions of neurons, and the intricate network of blood vessels with fluorescent labels in the brain. However, the current resin embedding method is inadequate because of incompatibilities with fluorescent dyes, making it difficult to reconstruct a variety of structures for the interpretation of their complex spatial relationships. We modified the resin embedding method for large biological tissues labeled with multiple fluorescent dyes and proteins through different labeling strategies. With TrueBlack as the background fluorescence inhibitor in the glycol methacrylate (GMA) embedding, we referred to the method as GMA-T (Glycol methacrylate with TB). In the GMA-T embedded mouse brains, structures labeled with fluorescent proteins and dyes were visualized in millimeter-scale networks with sub-cellular resolution, allowing quantitative analysis of different anatomical structures in the same brain, including neurons and blood vessels. In combination with high-resolution whole-brain imaging, it is possible to obtain a variety of fluorescence labeled structures in just a few days. We quantified the distribution and morphology of the tdTomato-labeled vasoactive intestinal polypeptide (VIP) neurons and the BSA-FITC labeled blood vessels in the same brain. These results demonstrated that VIP neurons and blood vessels have their own unique distribution patterns and morphological characteristics among cortical regions and different layers in cerebral cortex, and there was no significant correlation between VIP neurons and vessels. This approach provides a novel approach to study the interaction among different anatomical structures within large-volume biological samples labeled with multiple fluorescent dyes and proteins, which helps elucidating the complex anatomical characteristics of biological organs.

The Transcription Factor Ventral Anterior Homeobox 1 Modulates Circadian Time-Keeping and Fertility Through Direct Regulation of Vasoactive Intestinal Polypeptide Expression in the Suprachiasmatic Nucleus

Light provides the primary timing signal that enables fine-tuned behavioral and hormonal entrainment of circadian rhythms to the environment. Light is transmitted from the eye to the brain through the retinohypothalamic tract, where one target is the hypothalamic suprachiasmatic nucleus (SCN), which generates self-sustained circadian rhythms. The vasoactive intestinal polypeptide (VIP) expressing neurons of the SCN relay light information to peripheral cells and tissues through control of hormonal and nervous signals, allowing synchronization of molecular clocks located in individual cells throughout the body. Non-natural light cycles, ie shiftwork, and weakened SCN function through genetic manipulation, disrupt the body’s circadian rhythms, causing deregulated hormone release, metabolic disorders, and negative effects on reproductive systems such as irregular menstrual cycles and decreased sperm count. To further our understanding of how the SCN translates light information into neuroendocrine control of fertility, we conditionally deleted the SCN enriched transcription factor Ventral anterior homeobox 1 (Vax1) in post-developmental VIP neurons, generating Vax1-flox/flox:Vip-Cre+ (cKO) mice. To determine if the SCN timekeeping function was impacted in cKO mice, we single housed males and females with running wheels to examine activity during both 12-hour light/dark cycles and in constant darkness. Wheel-running behavior in constant darkness revealed a shortening of the endogenous free-running period (Tau) of the SCN. Aside from Tau, wheel running behaviors were comparable to controls. Weakened SCN output can negatively impact fertility. While on 12-hour light/dark cycles, we found a modest, but significant change in follicle stimulating hormone and estrogen in cKO females and a reduced sensitivity of GnRH neurons to kisspeptin in males. The changes in hormone release were associated with a slightly lengthened estrous cycle in cKO females and reduced sperm quality in cKO males. To identify the molecular origin of the shortened SCN period, we used immunohistochemistry and RNAscope to examine expression of Vip. We found that diestrus cKO females had a significant reduction in Vip expression at ZT16 and preliminary data suggest a reduction in the circadian clock gene Bmal1. Together, these studies identify a novel role of VAX1 in VIP neurons where VAX1 is required for VIP expression and circadian timekeeping. Loss of VAX1 in VIP neurons weakens SCN output, deregulating reproductive hormone release and modestly reducing reproductive function in both males and females.

Altered Light Sensitivity of Circadian Clock in Shank3+/- Mouse.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairment in communication and social interaction, repetitive or stereotypical behaviors, altered sensory perception, and sleep disorders. In general, the causes of ASD remain unknown, but in Phelan–McDermid syndrome, it is known that the disorder is related to the haploinsufficiency of the Shank3 gene. We used an autism model with compromised glutamatergic signaling, the Shank3+/– mouse, to study the circadian rhythm architecture of locomotion behavior and its entrainment to light. We also analyzed the synapse between the retinohypothalamic tract (RHT) and the suprachiasmatic nucleus (SCN), employing tract tracing and immunohistochemical techniques. We found that Shank3+/– mice were not impaired in the SCN circadian clock, as indicated by a lack of differences between groups in the circadian architecture in entrained animals to either long or short photoperiods. Circadian rhythm periodicity (tau) was unaltered between genotypes in constant darkness (DD, dim red light). Similar results were obtained in the re-entrainment to shifts in the light–dark cycle and in the entrainment to a skeleton photoperiod from DD. However, Shank3+/– mice showed larger phase responses to light pulses, both delays and advances, and rhythm disorganization induced by constant bright light. Immunohistochemical analyses indicated no differences in the RHT projection to the SCN or the number of SCN neurons expressing the N-methyl-D-aspartate (NMDA) receptor subunit NR2A, whereas the Shank3+/– animals showed decreased c-Fos induction by brief light pulses at CT14, but increased number of vasoactive intestinal polypeptide (VIP)-positive neurons. These results indicate alterations in light sensitivity in Shank3+/– mice. Further studies are necessary to understand the mechanisms involved in such increased light sensitivity, probably involving VIP neurons.

Dysregulation of epithelial ion transport and neurochemical changes in the colon of a parkinsonian primate

The pathological changes underlying gastrointestinal (GI) dysfunction in Parkinson’s disease (PD) are poorly understood and the symptoms remain inadequately treated. In this study we compared the functional and neurochemical changes in the enteric nervous system in the colon of adult, L-DOPA-responsive, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated common marmoset, with naïve controls. Measurement of mucosal vectorial ion transport, spontaneous longitudinal smooth muscle activity and immunohistochemical assessment of intrinsic innervation were each performed in discrete colonic regions of naïve and MPTP-treated marmosets. The basal short circuit current (Isc) was lower in MPTP-treated colonic mucosa while mucosal resistance was unchanged. There was no difference in basal cholinergic tone, however, there was an increased excitatory cholinergic response in MPTP-treated tissues when NOS was blocked with L-Nω-nitroarginine. The amplitude and frequency of spontaneous contractions in longitudinal smooth muscle as well as carbachol-evoked post-junctional contractile responses were unaltered, despite a decrease in choline acetyltransferase and an increase in the vasoactive intestinal polypeptide neuron numbers per ganglion in the proximal colon. There was a low-level inflammation in the proximal but not the distal colon accompanied by a change in α-synuclein immunoreactivity. This study suggests that MPTP treatment produces long-term alterations in colonic mucosal function associated with amplified muscarinic mucosal activity but decreased cholinergic innervation in myenteric plexi and increased nitrergic enteric neurotransmission. This suggests that long-term changes in either central or peripheral dopaminergic neurotransmission may lead to adaptive changes in colonic function resulting in alterations in ion transport across mucosal epithelia that may result in GI dysfunction in PD.

Increased Callosal Connectivity in Reeler Mice Revealed by Brain-Wide Input Mapping of VIP Neurons in Barrel Cortex.

The neocortex is composed of layers. Whether layers constitute an essential framework for the formation of functional circuits is not well understood. We investigated the brain-wide input connectivity of vasoactive intestinal polypeptide (VIP) expressing neurons in the reeler mouse. This mutant is characterized by a migration deficit of cortical neurons so that no layers are formed. Still, neurons retain their properties and reeler mice show little cognitive impairment. We focused on VIP neurons because they are known to receive strong long-range inputs and have a typical laminar bias toward upper layers. In reeler, these neurons are more dispersed across the cortex. We mapped the brain-wide inputs of VIP neurons in barrel cortex of wild-type and reeler mice with rabies virus tracing. Innervation by subcortical inputs was not altered in reeler, in contrast to the cortical circuitry. Numbers of long-range ipsilateral cortical inputs were reduced in reeler, while contralateral inputs were strongly increased. Reeler mice had more callosal projection neurons. Hence, the corpus callosum was larger in reeler as shown by structural imaging. We argue that, in the absence of cortical layers, circuits with subcortical structures are maintained but cortical neurons establish a different network that largely preserves cognitive functions.

Dual-Color Single-Cell Imaging of the Suprachiasmatic Nucleus Reveals a Circadian Role in Network Synchrony

The suprachiasmatic nucleus (SCN) acts as a master pacemaker driving circadian behavior and physiology. Although the SCN is small, it is composed of many cell types, making it difficult to study the roles of particular cells. Here we develop bioluminescent circadian reporter mice that are Cre dependent, allowing the circadian properties of genetically defined populations of cells to be studied in real time. Using a Color-Switch PER2::LUCIFERASE reporter that switches from red PER2::LUCIFERASE to green PER2::LUCIFERASE upon Cre recombination, we assess circadian rhythms in two of the major classes of peptidergic neurons in the SCN: AVP (arginine vasopressin) and VIP (vasoactive intestinal polypeptide). Surprisingly, we find that circadian function in AVP neurons, not VIP neurons, is essential for autonomous network synchrony of the SCN and stability of circadian rhythmicity.

Different Roles for VIP Neurons in the Neonatal and Adult Suprachiasmatic Nucleus.

The suprachiasmatic nucleus (SCN) drives circadian rhythms in locomotion through coupled, single-cell oscillations. Global genetic deletion of the neuropeptide Vip or its receptor Vipr2 results in profound deficits in daily synchrony among SCN cells and daily rhythms in locomotor behavior and glucocorticoid secretion. To test whether this phenotype depends on vasoactive intestinal polypeptide (VIP) neurons in the SCN, we ablated VIP SCN neurons in vivo in adult male mice through Caspase3-mediated induction of the apoptotic pathway in cre-expressing VIP neurons. We found that ablation of VIP SCN neurons in adult mice caused a phenotype distinct from Vip- and Vipr2-null mice. Mice lacking VIP neurons retained rhythmic locomotor activity with a shortened circadian period, more variable onsets, and decreased duration of daily activity. Circadian hormonal outputs, specifically corticosterone rhythms, were severely dampened. In contrast, deletion of neonatal SCN VIP neurons dramatically reduced circadian gene expression in the cultured SCN, mimicking the effects of global deletion of Vip or Vipr2. These results suggest that SCN VIP neurons play a role in lengthening circadian period and stimulating the daily surge in glucocorticoids in adults and in synchronizing and sustaining daily rhythms among cells in the developing SCN.

Reduced VIP Expression Affects Circadian Clock Function in VIP-IRES-CRE Mice (JAX 010908).

Circadian rhythms are programmed by the suprachiasmatic nucleus (SCN), which relies on neuropeptide signaling to maintain daily timekeeping. Vasoactive intestinal polypeptide (VIP) is critical for SCN function, but the precise role of VIP neurons in SCN circuits is not fully established. To interrogate their contribution to SCN circuits, VIP neurons can be manipulated specifically using the DNA-editing enzyme Cre recombinase. Although the Cre transgene is assumed to be inert by itself, we find that VIP expression is reduced in both heterozygous and homozygous adult VIP-IRES-Cre mice (JAX 010908). Compared with wild-type mice, homozygous VIP-Cre mice display faster reentrainment and shorter free-running period but do not become arrhythmic in constant darkness. Consistent with this phenotype, homozygous VIP-Cre mice display intact SCN PER2::LUC rhythms, albeit with altered period and network organization. We present evidence that the ability to sustain molecular rhythms in the VIP-Cre SCN is not due to residual VIP signaling; rather, arginine vasopressin signaling helps to sustain SCN function at both intracellular and intercellular levels in this model. This work establishes that the VIP-IRES-Cre transgene interferes with VIP expression but that loss of VIP can be mitigated by other neuropeptide signals to help sustain SCN function. Our findings have implications for studies employing this transgenic model and provide novel insight into neuropeptide signals that sustain daily timekeeping in the master clock.

Output from VIP cells of the mammalian central clock regulates daily physiological rhythms

The suprachiasmatic nucleus (SCN) circadian clock is critical for optimising daily cycles in mammalian physiology and behaviour. The roles of the various SCN cell types in communicating timing information to downstream physiological systems remain incompletely understood, however. In particular, while vasoactive intestinal polypeptide (VIP) signalling is essential for SCN function and whole animal circadian rhythmicity, the specific contributions of VIP cell output to physiological control remains uncertain. Here we reveal a key role for SCN VIP cells in central clock output. Using multielectrode recording and optogenetic manipulations, we show that VIP neurons provide coordinated daily waves of GABAergic input to target cells across the paraventricular hypothalamus and ventral thalamus, supressing their activity during the mid to late day. Using chemogenetic manipulation, we further demonstrate specific roles for this circuitry in the daily control of heart rate and corticosterone secretion, collectively establishing SCN VIP cells as influential regulators of physiological timing.

Characterization of Oxytocin Receptor Expression Within Various Neuronal Populations of the Mouse Dorsal Hippocampus.

Oxytocin, acting through the oxytocin receptor (Oxtr) in the periphery, is best known for its roles in regulating parturition and lactation. However, it is also now known to possess a number of important social functions within the central nervous system, including social preference, memory and aggression, that vary to different degrees in different species. The Oxtr is found in both excitatory and inhibitory neurons within the brain and research is focusing on how, for example, activation of the receptor in interneurons can enhance the signal-to-noise of neuronal transmission. It is important to understand which neurons in the mouse dorsal hippocampus might be activated during memory formation. Therefore, we examined the colocalization of transcripts in over 5,000 neurons for Oxtr with those for nine different markers often found in interneurons using hairpin chain reaction in situ hybridization on hippocampal sections. Most pyramidal cell neurons of CA2 and many in the CA3 express Oxtr. Outside of those excitatory neurons, over 90% of Oxtr-expressing neurons co-express glutamic acid decarboxylase-1 (Gad-1) with progressively decreasing numbers of co-expressing cholecystokinin, somatostatin, parvalbumin, neuronal nitric oxide synthase, the serotonin 3a receptor, the vesicular glutamate transporter 3, calbindin 2 (calretinin), and vasoactive intestinal polypeptide neurons. Distributions were analyzed within hippocampal layers and regions as well. These findings indicate that Oxtr activation will modulate the activity of ~30% of the Gad-1 interneurons and the majority of the diverse population of those, mostly, interneuron types specifically examined in the mouse hippocampus.

Neuromodulation Leads to a Burst-Tonic Switch in a Subset of VIP Neurons in Mouse Primary Somatosensory (Barrel) Cortex.

Neocortical GABAergic interneurons expressing vasoactive intestinal polypeptide (VIP) contribute to sensory processing, sensorimotor integration, and behavioral control. In contrast to other major subpopulations of GABAergic interneurons, VIP neurons show a remarkable diversity. Studying morphological and electrophysiological properties of VIP cells, we found a peculiar group of neurons in layer II/III of mouse primary somatosensory (barrel) cortex, which showed a highly dynamic burst firing behavior at resting membrane potential that switched to tonic mode at depolarized membrane potentials. Furthermore, we demonstrate that burst firing depends on T-type calcium channels. The burst-tonic switch could be induced by acetylcholine (ACh) and serotonin. ACh mediated a depolarization via nicotinic receptors whereas serotonin evoked a biphasic depolarization via ionotropic and metabotropic receptors in 48% of the population and a purely monophasic depolarization via metabotropic receptors in the remaining cells. These data disclose an electrophysiologically defined subpopulation of VIP neurons that via neuromodulator-induced changes in firing behavior is likely to regulate the state of cortical circuits in a profound manner.

Whole-Brain Monosynaptic Afferent Projections to the Cholecystokinin Neurons of the Suprachiasmatic Nucleus.

The suprachiasmatic nucleus (SCN) is the principal pacemaker driving the circadian rhythms of physiological behaviors. The SCN consists of distinct neurons expressing neuropeptides, including arginine vasopressin (AVP), vasoactive intestinal polypeptide (VIP), gastrin-releasing peptide (GRP), cholecystokinin (CCK), and so on. AVP, VIP, and GRP neurons receive light stimulation from the retina to synchronize endogenous circadian clocks with the solar day, whereas CCK neurons are not directly innervated by retinal ganglion cells and may be involved in the non-photic regulation of the circadian clock. To better understand the function of CCK neurons in non-photic circadian rhythm, it is vital to clarify the direct afferent inputs to CCK neurons in the SCN. Here, we utilized a recently developed rabies virus- and Cre/loxP-based, cell type-specific, retrograde tracing system to map and quantitatively analyze the whole-brain monosynaptic inputs to SCN CCK neurons. We found that SCN CCK neurons received direct inputs from 29 brain nuclei. Among these nuclei, paraventricular nucleus of the hypothalamus (PVH), paraventricular nucleus of the thalamus (PVT), supraoptic nucleus (SON), ventromedial nucleus of the hypothalamus, and seven other nuclei sent numerous inputs to CCK neurons. Moderate inputs originated from the zona incerta, periventricular hypothalamic nucleus, and five other nuclei. A few inputs to CCK neurons originated from the orbital frontal cortex, prelimbic cortex, cingulate cortex, claustrum, and seven other nuclei. In addition, SCN CCK neurons were preferentially innervated by AVP neurons of the ipsilateral PVH and SON rather than their contralateral counterpart, whereas the contralateral PVT sent more projections to CCK neurons than to its ipsilateral counterpart. Taken together, these results expand our knowledge of the specific innervation to mouse SCN CCK neurons and provide an important indication for further investigations on the function of CCK neurons.

Connectome of the Suprachiasmatic Nucleus: New Evidence of the Core-Shell Relationship.

A brain clock, constituted of ∼20,000 peptidergically heterogeneous neurons, is located in the hypothalamic suprachiasmatic nucleus (SCN). While many peptidergic cell types have been identified, little is known about the connections among these neurons in mice. We first sought to identify contacts among major peptidergic cell types in the SCN using triple-label fluorescent immunocytochemistry (ICC). To this end, contacts among vasoactive intestinal polypeptide (VIP), gastrin-releasing peptide (GRP), and calretinin (CALR) cells of the core, and arginine vasopressin (AVP) and met-enkephalin (ENK) cells of the shell were analyzed. Some core-to-shell and shell-to-core communications are specialized. We found that in wild-type (WT) mice, AVP fibers make extremely sparse contacts onto VIP neurons but contacts in the reverse direction are numerous. In contrast, AVP fibers make more contacts onto GRP neurons than conversely. For the other cell types tested, largely reciprocal connections are made. These results point to peptidergic cell type-specific communications between core and shell SCN neurons. To further understand the impact of VIP-to-AVP communication, we next explored the SCN in VIP-deficient mice (VIP-KO). In these animals, AVP expression is markedly reduced in the SCN, but it is not altered in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). Surprisingly, in VIP-KO mice, the number of AVP appositions onto other peptidergic cell types is not different from controls. Colchicine administration, which blocks AVP transport, restored the numbers of AVP neurons in VIP-KO to that of WT littermates. The results indicate that VIP has an important role in modulating AVP expression levels in the SCN in this mouse.