Involvement of Denervated Midbrain-Derived Factors in the Formation of Ectopic Cortico-Mesencephalic Projection after Hemispherectomy.

Neuronal remodeling after brain injury is essential for functional recovery. After unilateral cortical lesion, axons from the intact cortex ectopically project to the denervated midbrain, but the molecular mechanisms remain largely unknown. To address this issue, we examined gene expression profiles in denervated and intact mouse midbrains after hemispherectomy at early developmental stages using mice of either sex, when ectopic contralateral projection occurs robustly. The analysis showed that various axon growth-related genes were upregulated in the denervated midbrain, and most of these genes are reportedly expressed by glial cells. To identify the underlying molecules, the receptors for candidate upregulated molecules were knocked out in layer 5 projection neurons in the intact cortex, using the CRISPR/Cas9-mediated method, and axonal projection from the knocked-out cortical neurons was examined after hemispherectomy. We found that the ectopic projection was significantly reduced when integrin subunit ß three or neurotrophic receptor tyrosine kinase 2 (also known as TrkB) was knocked out. Overall, the present study suggests that denervated midbrain-derived glial factors contribute to lesion-induced remodeling of the cortico-mesencephalic projection via these receptors.SIGNIFICANCE STATEMENT After brain injury, compensatory neural circuits are established that contribute to functional recovery. However, little is known about the intrinsic mechanism that underlies the injury-induced remodeling. We found that after unilateral cortical ablation expression of axon-growth promoting factors is elevated in the denervated midbrain and is involved in the formation of ectopic axonal projection from the intact cortex. Evidence further demonstrated that these factors are expressed by astrocytes and microglia, which are activated in the denervated midbrain. Thus, our present study provides a new insight into the mechanism of lesion-induced axonal remodeling and further therapeutic strategies after brain injury.

Hippocampal and thalamic afferents form distinct synaptic microcircuits in the mouse infralimbic frontal cortex.

The selection of goal-directed behaviors is supported by neural circuits located within the frontal cortex. Frontal cortical afferents arise from multiple brain areas, yet the cell-type-specific targeting of these inputs is unclear. Here, we use monosynaptic retrograde rabies mapping to examine the distribution of afferent neurons targeting distinct classes of local inhibitory interneurons and excitatory projection neurons in mouse infralimbic frontal cortex. Interneurons expressing parvalbumin, somatostatin, or vasoactive intestinal peptide receive a large proportion of inputs from the hippocampus, while interneurons expressing neuron-derived neurotrophic factor receive a large proportion of inputs from thalamic regions. A similar dichotomy is present among the four different excitatory projection neurons. These results show a prominent bias among long-range hippocampal and thalamic afferent systems in their targeting to specific sets of frontal cortical neurons. Moreover, they suggest the presence of two distinct local microcircuits that control how different inputs govern frontal cortical information processing.

Cell-type-specific recruitment of GABAergic interneurons in the primary somatosensory cortex by long-range inputs.

Extensive hierarchical yet highly reciprocal interactions among cortical areas are fundamental for information processing. However, connectivity rules governing the specificity of such corticocortical connections, and top-down feedback projections in particular, are poorly understood. We analyze synaptic strength from functionally relevant brain areas to diverse neuronal types in the primary somatosensory cortex (S1). Long-range projections from different areas preferentially engage specific sets of GABAergic neurons in S1. Projections from other somatosensory cortices strongly recruit parvalbumin (PV)-positive GABAergic neurons and lead to PV neuron-mediated feedforward inhibition of pyramidal neurons in S1. In contrast, inputs from whisker-related primary motor cortex are biased to vasoactive intestinal peptide (VIP)-positive GABAergic neurons and potentially result in VIP neuron-mediated disinhibition. Regardless of the input areas, somatostatin-positive neurons receive relatively weak long-range inputs. Computational analyses suggest that a characteristic combination of synaptic inputs to different GABAergic IN types in S1 represents a specific long-range input area.

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.

Exclusive labeling of direct and indirect pathway neurons in the mouse neostriatum by an adeno-associated virus vector with Cre/lox system.

We developed an adeno-associated virus (AAV) vector-based technique to label mouse neostriatal neurons comprising direct and indirect pathways with different fluorescent proteins and analyze their axonal projections. The AAV vector expresses GFP or RFP in the presence or absence of Cre recombinase and should be useful for labeling two cell populations exclusively dependent on its expression. Here, we describe the AAV vector design, stereotaxic injection of the AAV vector, and a highly sensitive immunoperoxidase method for axon visualization. For complete details on the use and execution of this protocol, please refer to Okamoto et al. (2020).

Activation of the ARCPOMC→MeA Projection Reduces Food Intake.

Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC) plays an essential role in the control of food intake and energy expenditure. Melanocortin-4 receptors (MC4Rs) are expressed in key areas that are implicated in regulating energy homeostasis. Although the importance of MC4Rs in the paraventricular hypothalamus (PVH) has been well documented, the role of MC4Rs in the medial amygdala (MeA) on feeding remains controversial. In this study, we specifically examine the role of a novel ARCPOMC→MeA neural circuit in the regulation of short-term food intake. To map a local melanocortinergic neural circuit, we use monosynaptic anterograde as well as retrograde viral tracers and perform double immunohistochemistry to determine the identity of the neurons receiving synaptic input from POMC neurons in the ARC. To investigate the role of the ARCPOMC→MeA projection on feeding, we optogenetically stimulate channelrhodopsin-2 (ChR2)-expressing POMC fibers in the MeA. Anterograde viral tracing studies reveal that ARC POMC neurons send axonal projections to estrogen receptor-α (ER-α)- and MC4R-expressing neurons in the MeA. Retrograde viral tracing experiments show that the neurons projecting to the MeA is located mainly in the lateral part of the ARC. Optogenetic stimulation of the ARCPOMC→MeA pathway reduces short-term food intake. This anorectic effect is blocked by treatment with the MC4R antagonist SHU9119. In addition to the melanocortinergic local circuits within the hypothalamus, this extrahypothalamic ARCPOMC→MeA neural circuit would play a role in regulating short-term food intake.

Cellular Expression and Functional Roles of All 26 Neurotransmitter GPCRs in the C. elegans Egg-Laying Circuit.

Maps of the synapses made and neurotransmitters released by all neurons in model systems, such as Caenorhabditis elegans have left still unresolved how neural circuits integrate and respond to neurotransmitter signals. Using the egg-laying circuit of C. elegans as a model, we mapped which cells express each of the 26 neurotransmitter GPCRs of this organism and also genetically analyzed the functions of all 26 GPCRs. We found that individual neurons express many distinct receptors, epithelial cells often express neurotransmitter receptors, and receptors are often positioned to receive extrasynaptic signals. Receptor knockouts reveal few egg-laying defects under standard laboratory conditions, suggesting that the receptors function redundantly or regulate egg-laying only in specific conditions; however, increasing receptor signaling through overexpression more efficiently reveals receptor functions. This map of neurotransmitter GPCR expression and function in the egg-laying circuit provides a model for understanding GPCR signaling in other neural circuits.SIGNIFICANCE STATEMENT Neurotransmitters signal through GPCRs to modulate activity of neurons, and changes in such signaling can underlie conditions such as depression and Parkinson's disease. To determine how neurotransmitter GPCRs together help regulate function of a neural circuit, we analyzed the simple egg-laying circuit in the model organism C. elegans We identified all the cells that express every neurotransmitter GPCR and genetically analyzed how each GPCR affects the behavior the circuit produces. We found that many neurotransmitter GPCRs are expressed in each neuron, that neurons also appear to use these receptors to communicate with other cell types, and that GPCRs appear to often act redundantly or only under specific conditions to regulate circuit function.

Posterior amygdala regulates sexual and aggressive behaviors in male mice.

Sexual and aggressive behaviors are fundamental to animal survival and reproduction. The medial preoptic nucleus (MPN) and ventrolateral part of the ventromedial hypothalamus (VMHvl) are essential regions for male sexual and aggressive behaviors, respectively. While key inhibitory inputs to the VMHvl and MPN have been identified, the extrahypothalamic excitatory inputs essential for social behaviors remain elusive. Here we identify estrogen receptor alpha (Esr1)-expressing cells in the posterior amygdala (PA) as a main source of excitatory inputs to the hypothalamus and key mediators for mating and fighting in male mice. We find two largely distinct PA subpopulations that differ in connectivity, gene expression, in vivo responses and social behavior relevance. MPN-projecting PAEsr1+ cells are activated during mating and are necessary and sufficient for male sexual behaviors, while VMHvl-projecting PAEsr1+ cells are excited during intermale aggression and promote attacks. These findings place the PA as a key node in both male aggression and reproduction circuits.

Direct Parabrachial-Cortical Connectivity.

The parabrachial nucleus (PB) in the upper brain stem tegmentum includes several neuronal subpopulations with a wide variety of connections and functions. A subpopulation of PB neurons projects axons directly to the cerebral cortex, and limbic areas of the cerebral cortex send a return projection directly to the PB. We used retrograde and Cre-dependent anterograde tracing to identify genetic markers and characterize this PB-cortical interconnectivity in mice. Cortical projections originate from glutamatergic PB neurons that contain Lmx1b (81%), estrogen receptor alpha (26%), and Satb2 (20%), plus mRNA for the neuropeptides cholecystokinin (Cck, 48%) and calcitonin gene-related peptide (Calca, 13%), with minimal contribution from FoxP2+ PB neurons (2%). Axons from the PB produce an extensive terminal field in an unmyelinated region of the insular cortex, extending caudally into the entorhinal cortex, and arcing rostrally through the dorsolateral prefrontal cortex, with a secondary terminal field in the medial prefrontal cortex. In return, layer 5 neurons in the insular cortex and other prefrontal areas, along with a dense cluster of cells dorsal to the claustrum, send a descending projection to subregions of the PB that contain cortically projecting neurons. This information forms the neuroanatomical basis for testing PB-cortical interconnectivity in arousal and interoception.

Neural connectivity between the hypothalamic supramammillary nucleus and appetite- and motivation-related regions of the rat brain.

The supramammillary nucleus (SuM) has an emerging role in appetite control. We have shown that the rat SuM is activated during hunger or food anticipation, or by ghrelin administration. In the present study, we characterised the connectivity between the SuM and key appetite- and motivation-related nuclei in the rat. In adult wild-type rats, or rats expressing Cre recombinase under the control of the tyrosine hydroxylase (TH) promoter (TH-Cre rats), we used c-Fos immunohistochemistry to visualise and correlate the activation of medial SuM (SuMM) with activation in the lateral hypothalamic area (LH), the dorsomedial hypothalamus (DMH) or the ventral tegmental area (VTA) after voluntary consumption of a high-sugar, high-fat food. To determine neuroanatomical connectivity, we used retrograde and anterograde tracing methods to specifically investigate the neuronal inputs and outputs of the SuMM. After consumption of the food there were positive correlations between c-Fos expression in the SuMM and the LH, DMH and VTA (P = 0.0001, 0.01 and 0.004). Using Fluoro-Ruby as a retrograde tracer, we demonstrate the existence of inputs from the LH, DMH, VTA and ventromedial hypothalamus (VMH) to the SuMM. The SuMM showed reciprocal inputs to the LH and DMH, and we identified a TH-positive output from SuMM to DMH. We co-labelled retrogradely-labelled sections for TH in the VMH, or for TH, orexin and melanin-concentrating hormone in the LH and DMH. However, we did not observe any colocalisation of immunoreactivity with any retrogradely-labelled cells. Viral mapping in TH-Cre rats confirms the existence of a reciprocal SuMM-DMH connection and shows that TH-positive cells project from the SuMM and VTA to the lateral septal area and cingulate cortex, respectively. These data provide evidence for the connectivity of the SuMM to brain regions involved in appetite control, and form the foundation for functional and behavioural studies aiming to further characterise the brain circuitry controlling eating behaviours.

Candidates for photic entrainment pathways to the circadian clock via optic lobe neuropils in the Madeira cockroach.

The compound eye of cockroaches is obligatory for entrainment of the Madeira cockroach's circadian clock, but the cellular nature of its entrainment pathways is enigmatic. Employing multiple-label immunocytochemistry, histochemistry, and backfills, we searched for photic entrainment pathways to the accessory medulla (AME), the circadian clock of the Madeira cockroach. We wanted to know whether photoreceptor terminals could directly contact pigment-dispersing factor-immunoreactive (PDF-ir) circadian pacemaker neurons with somata in the lamina (PDFLAs) or somata next to the AME (PDFMEs). Short green-sensitive photoreceptor neurons of the compound eye terminated in lamina layers LA1 and LA2, adjacent to PDFLAs and PDFMEs that branched in LA3. Long UV-sensitive compound eye photoreceptor neurons terminated in medulla layer ME2 without direct contact to ipsilateral PDFMEs that arborized in ME4. Multiple neuropeptide-ir interneurons branched in ME4, connecting the AME to ME2. Before, extraocular photoreceptors of the lamina organ were suggested to send terminals to accessory laminae. There, they overlapped with PDFLAs that mostly colocalized PDF, FMRFamide, and 5-HT immunoreactivities, and with terminals of ipsi- and contralateral PDFMEs. We hypothesize that during the day cholinergic activation of the largest PDFME via lamina organ photoreceptors maintains PDF release orchestrating phases of sleep-wake cycles. As ipsilateral PDFMEs express excitatory and contralateral PDFMEs inhibitory PDF autoreceptors, diurnal PDF release keeps both PDF-dependent clock circuits in antiphase. Future experiments will test whether ipsilateral PDFMEs are sleep-promoting morning cells, while contralateral PDFMEs are activity-promoting evening cells, maintaining stable antiphase via the largest PDFME entrained by extraocular photoreceptors of the lamina organ.

Maturation of thalamocortical synapses in the somatosensory cortex depends on neocortical AKAP5 expression.

Sensory cortex topographic maps consist of organized arrays of thalamocortical afferents (TCAs) that project into distinct areas of the cortex. Formation of topographic maps in sensory cortices is a prerequisite for functional maturation of the neocortex. Studies have shown that the formation of topographic maps and the maturation of thalamocortical synapses in the somatosensory cortex depend on the cyclic adenosine 5'-monophosphate-(cAMP)-protein kinase A (PKA) signaling pathway. AKAP5 is a scaffold protein (also called AKAP79 in humans or AKAP150 in rodents; AKAP79/150) that serves as a signaling hub that links cAMP and PKA signaling. Whether AKAP5 plays a role in topographic map formation and the maturation of thalamocortical synapses during development of the somatosensory cortex is still unknown. Here, we generated cortex-specific AKAP5-knockout mice (CxAKAP5KO) to examine its roles in somatosensory cortex development. We found that CxAKAP5KO mice displayed impaired cortical barrel maps. Electrophysiological recordings showed that the AMPA/NMDA ratio was reduced, and silent synapses were increased in thalamocortical synapses of CxAKAP5KO mice during postnatal development. Morphological analysis of layer IV cortical neurons demonstrated that dendritic refinement of these neurons was abnormal. These results indicate that AKAP5 is necessary for both topographic map formation and maturation of thalamocortical synapses as well as morphological development of cortical neurons in the somatosensory cortex.

Evolution of Brain Connections: Integrating Diffusion MR Tractography With Gene Expression Highlights Increased Corticocortical Projections in Primates.

Diffusion MR tractography permits investigating the 3D structure of cortical pathways as interwoven paths across the entire brain. We use high-resolution scans from diffusion spectrum imaging and high angular resolution diffusion imaging to investigate the evolution of cortical pathways within the euarchontoglire (i.e., primates, rodents) lineage. More specifically, we compare cortical fiber pathways between macaques (Macaca mulatta), marmosets (Callithrix jachus), and rodents (mice, Mus musculus). We integrate these observations with comparative analyses of Neurofilament heavy polypeptide (NEFH) expression across the cortex of mice and primates. We chose these species because their phylogenetic position serves to trace the early evolutionary history of the human brain. Our comparative analysis from diffusion MR tractography, cortical white matter scaling, and NEFH expression demonstrates that the examined primates deviate from mice in possessing increased long-range cross-cortical projections, many of which course across the anterior to posterior axis of the cortex. Our study shows that integrating gene expression data with diffusion MR data is an effective approach in identifying variation in connectivity patterns between species. The expansion of corticocortical pathways and increased anterior to posterior cortical integration can be traced back to an extension of neurogenetic schedules during development in primates.

Mapping neuronal inputs to Kiss1 neurons in the arcuate nucleus of the mouse.

The normal function of the mammalian reproductive axis is strongly influenced by physiological, metabolic and environmental factors. Kisspeptin neuropeptides, encoded by the Kiss1 gene, are potent regulators of the mammalian reproductive axis by stimulating gonadodropin releasing hormone secretion from the hypothalamus. To understand how the reproductive axis is modulated by higher order neuronal inputs we have mapped the afferent circuits into arcuate (ARC) Kiss1 neurons. We used a transgenic mouse that expresses the CRE recombinase in Kiss1 neurons for conditional viral tracing with genetically modified viruses. CRE-mediated activation of these viruses in Kiss1 neurons allows the virus to move transynaptically to label neurons with primary or secondary afferent inputs into the Kiss1 neurons. Several regions of the brain showed synaptic connectivity to arcuate Kiss1 neurons including proopiomelanocortin neurons in the ARC itself, kisspeptin neurons in the anteroventral periventricular nucleus, vasopressin neurons in the supraoptic and suprachiasmatic nuclei, thyrotropin releasing neurons in the paraventricular nucleus and unidentified neurons in other regions including the subfornical organ, amygdala, interpeduncular nucleus, ventral premammilary nucleus, basal nucleus of stria terminalis and the visual, somatosensory and piriform regions of the cortex. These data provide an insight into how the activity of Kiss1 neurons may be regulated by metabolic signals and provide a detailed neuroanatomical map for future functional studies.

New viral-genetic mapping uncovers an enrichment of corticotropin-releasing hormone-expressing neuronal inputs to the nucleus accumbens from stress-related brain regions.

Corticotropin-releasing hormone (CRH) is an essential, evolutionarily-conserved stress neuropeptide. In addition to hypothalamus, CRH is expressed in brain regions including amygdala and hippocampus where it plays crucial roles in modulating the function of circuits underlying emotion and cognition. CRH+ fibers are found in nucleus accumbens (NAc), where CRH modulates reward/motivation behaviors. CRH actions in NAc may vary by the individual's stress history, suggesting roles for CRH in neuroplasticity and adaptation of the reward circuitry. However, the origin and extent of CRH+ inputs to NAc are incompletely understood. We employed viral genetic approaches to map both global and CRH+ projection sources to NAc in mice. We injected into NAc variants of a new designer adeno-associated virus that permits robust retrograde access to NAc-afferent projection neurons. Cre-dependent viruses injected into CRH-Cre mice enabled selective mapping of CRH+ afferents. We employed anterograde AAV1-directed axonal tracing to verify NAc CRH+ fiber projections and established the identity of genetic reporter-labeled cells via validated antisera against native CRH. We quantified the relative contribution of CRH+ neurons to total NAc-directed projections. Combined retrograde and anterograde tracing identified the paraventricular nucleus of the thalamus, bed nucleus of stria terminalis, basolateral amygdala, and medial prefrontal cortex as principal sources of CRH+ projections to NAc. CRH+ NAc afferents were selectively enriched in NAc-projecting brain regions involved in diverse aspects of the sensing, processing and memory of emotionally salient events. These findings suggest multiple, complex potential roles for the molecularly-defined, CRH-dependent circuit in modulation of reward and motivation behaviors.

Neurons expressing estrogen receptor α differentially innervate the periaqueductal gray matter of female rats.

The periaqueductal gray matter (PAG) is a brainstem site involved in distinct autonomic and behavioral responses. Among them, the motor control of female sexual behavior, including lordosis, is well described. Lordosis reflex is highly dependent on increasing levels of estradiol that occur in the afternoon of the proestrus day in normally cycling females. This effect is thought to be mediated primarily via actions in the ventromedial nucleus of the hypothalamus (VMH). By binding to estrogen receptor α (ERα), estradiol changes the activity of VMH neurons that project to the PAG. Evidence also exists for the coordination of PAG outputs by estradiol-responsive neurons outside the VMH. However, a comprehensive analysis of these circuitries is not available. Using stereotaxic injection of the retrograde tracer Fluorogold in distinct columns of the PAG we performed a systematic mapping of neurons innervating the PAG and those coexpressing ERα immunoreactivity. We found that the forebrain projections to PAG columns are largely segregated and that most of the ERα expressing neurons preferentially target the lateral and the ventrolateral columns. Dual labeled neurons were mostly found in the intermediate subdivision of the lateral septal nucleus, the posterior aspect of the medial bed nucleus of the stria terminalis, the medial preoptic nucleus, the striohypothalamic nucleus and the ventrolateral VMH. Few dual labeled neurons were also observed in the arcuate nucleus, in the posterodorsal subdivision of the medial nucleus of the amygdala and in the ventral premammillary nucleus. Our findings indicate that ERα modulates sexual behavior in female rats via an integrated neural network that differentially innervate the columns of the PAG.

Morphological analysis for neuronal pathway from the hindbrain ependymocytes to the hypothalamic kisspeptin neurons.

Hindbrain ependymocytes are postulated to have a glucose-sensing role in regulating gonadal functions. Previous studies have suggested that malnutrition-induced suppression of gonadotropin secretion is mediated by noradrenergic inputs from the A2 region in the solitary tract nucleus to the paraventricular nucleus (PVN), and by corticotropin-releasing hormone (CRH) release in the hypothalamus. However, no morphological evidence to indicate the neural pathway from the hindbrain ependymocytes to hypothalamic kisspeptin neurons, a center for reproductive function in mammals, currently exists. The present study aimed to examine the existence of a neuronal pathway from the hindbrain ependymocytes to kisspeptin neurons in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). To determine this, wheat-germ agglutinin (WGA), a trans-synaptic tracer, was injected into the fourth ventricle (4V) in heterozygous Kiss1-tandem dimer Tomato (tdTomato) rats, where kisspeptin neurons were visualized by tdTomato fluorescence. 48 h after the WGA injection, brain sections were taken from the forebrain, midbrain and hindbrain and subjected to double immunohistochemistry for WGA and dopamine ß-hydroxylase (DBH) or CRH. WGA immunoreactivities were found in vimentin-immunopositive ependymocytes of the 4V and the central canal (CC), but not in the third ventricle. The WGA immunoreactivities were detected in some tdTomato-expressing cells in the ARC and AVPV, DBH-immunopositive cells in the A1-A7 noradrenergic nuclei, and CRH-immunopositive cells in the PVN. These results suggest that the hindbrain ependymocytes have neuronal connections with the kisspeptin neurons, most probably via hindbrain noradrenergic and CRH neurons to relay low energetic signals for regulation of reproduction.

GLP-1 modulates the supramammillary nucleus-lateral hypothalamic neurocircuit to control ingestive and motivated behavior in a sex divergent manner.

OBJECTIVE: The supramammillary nucleus (SuM) is nestled between the lateral hypothalamus (LH) and the ventral tegmental area (VTA). This neuroanatomical position is consistent with a potential role of this nucleus to regulate ingestive and motivated behavior. Here neuroanatomical, molecular, and behavior approaches are utilized to determine whether SuM contributes to ingestive and food-motivated behavior control. METHODS: Through the application of anterograde and retrograde neural tract tracing with novel designer viral vectors, the current findings show that SuM neurons densely innervate the LH in a sex dimorphic fashion. Glucagon-like peptide-1 (GLP-1) is a clinically targeted neuro-intestinal hormone with a well-established role in regulating energy balance and reward behaviors. Here we determine that GLP-1 receptors (GLP-1R) are expressed throughout the SuM of both sexes, and also directly on SuM LH-projecting neurons and investigate the role of SuM GLP-1R in the regulation of ingestive and motivated behavior in male and female rats. RESULTS: SuM microinjections of the GLP-1 analogue, exendin-4, reduced ad libitum intake of chow, fat, or sugar solution in both male and female rats, while food-motivated behaviors, measured using the sucrose motivated operant conditioning test, was only reduced in male rats. These data contrasted with the results obtained from a neighboring structure well known for its role in motivation and reward, the VTA, where females displayed a more potent response to GLP-1R activation by exendin-4. In order to determine the physiological role of SuM GLP-1R signaling regulation of energy balance, we utilized an adeno-associated viral vector to site-specifically deliver shRNA for the GLP-1R to the SuM. Surprisingly, and in contrast to previous results for the two SuM neighboring sites, LH and VTA, SuM GLP-1R knockdown increased food seeking and adiposity in obese male rats without altering food intake, body weight or food motivation in lean or obese, female or male rats. CONCLUSION: Taken together, these results indicate that SuM potently contributes to ingestive and motivated behavior control; an effect contingent on sex, diet/homeostatic energy balance state and behavior of interest. These data also extend the map of brain sites directly responsive to GLP-1 agonists, and highlight key differences in the role that GLP-1R play in interconnected and neighboring nuclei.

Input-output organization of the mouse claustrum.

Progress in determining the precise organization and function of the claustrum (CLA) has been hindered by the difficulty in reliably targeting these neurons. To overcome this, we used a projection-based targeting strategy to selectively label CLA principal neurons. Combined with adeno-associated virus (AAV) and monosynaptic rabies tracing techniques, we systematically examined the pre-synaptic input and axonal output of this structure. We found that CLA neurons projecting to retrosplenial cortex (RSP) collateralize extensively to innervate a variety of higher-order cortical regions. No subcortical labeling was found, with the exception of sparse terminals in the basolateral amygdala (BLA). This pattern of output was similar to cingulate- and visual cortex-projecting CLA neurons, suggesting a common targeting scheme among these projection-defined populations. Rabies virus tracing directly demonstrated widespread synaptic inputs to RSP-projecting CLA neurons from both cortical and subcortical areas. The strongest inputs arose from classically defined limbic regions, including medial prefrontal cortex, anterior cingulate, BLA, ventral hippocampus, and neuromodulatory systems such as the dorsal raphe and cholinergic basal forebrain. These results suggest that the CLA may integrate information related to the emotional salience of stimuli and may globally modulate cortical state by broadcasting its output uniformly across a variety of higher cognitive centers.

A characterization of laminar architecture in mouse primary auditory cortex.

Laminar architecture of primary auditory cortex (A1) has long been investigated by traditional histochemical techniques such as Nissl staining, retrograde and anterograde tracings. Uncertainty still remains, however, about laminar boundaries in mice. Here we investigated the cortical lamina structure by combining neuronal tracing and immunofluorochemistry for laminar specific markers. Most retrogradely labeled corticothalamic neurons expressed Forkhead box protein P2 (Foxp2) and distributed within the laminar band of Foxp2-expressing cells, identifying layer 6. Cut-like homeobox 1 (Cux1) expression in layer 2-4 neurons divided the upper layers into low expression layers 2/3 and high expression layers 3/4, which overlapped with the dense terminals of vesicular glutamate transporter 2 (vGluT2) and anterogradely labeled lemniscal thalamocortical axons. In layer 5, between Cux1-expressing layers 2-4 and Foxp2-defined layer 6, retrogradely labeled corticocollicular projection neurons mostly expressed COUP-TF interacting protein 2 (Ctip2). Ctip2-expressing neurons formed a laminar band in the middle of layer 5 distant from layer 6, creating a laminar gap between the two laminas. This gap contained a high population of commissural neurons projecting to contralateral A1 compared to other layers and received vGluT2-immunopositive, presumptive thalamocortical axon collateral inputs. Our study shows that layer 5 is much wider than layer 6, and layer 5 can be divided into at least three sublayers. The thalamorecipient layers 3/4 may be separated from layers 2/3 using Cux1 and can be also divided into layer 4 and layer 3 based on the neuronal soma size. These data provide a new insight for the laminar structure of mouse A1.