Ventral pallidum regulates the default mode network, controlling transitions between internally and externally guided behavior.

Daily life requires transitions between performance of well-practiced, automatized behaviors reliant upon internalized representations and behaviors requiring external focus. Such transitions involve differential activation of the default mode network (DMN), a group of brain areas associated with inward focus. We asked how optogenetic modulation of the ventral pallidum (VP), a subcortical DMN node, impacts task switching between internally to externally guided lever-pressing behavior in the rat. Excitation of the VP dramatically compromised acquisition of an auditory discrimination task, trapping animals in a DMN state of automatized internally focused behavior and impairing their ability to direct attention to external sensory stimuli. VP inhibition, on the other hand, facilitated task acquisition, expediting escape from the DMN brain state, thereby allowing rats to incorporate the contingency changes associated with the auditory stimuli. We suggest that VP, instant by instant, regulates the DMN and plays a deterministic role in transitions between internally and externally guided behaviors.

Basal forebrain contributes to default mode network regulation.

The default mode network (DMN) is a collection of cortical brain regions that is active during states of rest or quiet wakefulness in humans and other mammalian species. A pertinent characteristic of the DMN is a suppression of local field potential gamma activity during cognitive task performance as well as during engagement with external sensory stimuli. Conversely, gamma activity is elevated in the DMN during rest. Here, we document that the rat basal forebrain (BF) exhibits the same pattern of responses, namely pronounced gamma oscillations during quiet wakefulness in the home cage and suppression of this activity during active exploration of an unfamiliar environment. We show that gamma oscillations are localized to the BF and that gamma-band activity in the BF has a directional influence on a hub of the rat DMN, the anterior cingulate cortex, during DMN-dominated brain states. The BF is well known as an ascending, activating, neuromodulatory system involved in wake-sleep regulation, memory formation, and regulation of sensory information processing. Our findings suggest a hitherto undocumented role of the BF as a subcortical node of the DMN, which we speculate may be important for switching between internally and externally directed brain states. We discuss potential BF projection circuits that could underlie its role in DMN regulation and highlight that certain BF nuclei may provide potential target regions for up- or down-regulation of DMN activity that might prove useful for treatment of DMN dysfunction in conditions such as epilepsy or major depressive disorder.

Stress Impacts the Regulation Neuropeptides in the Rat Hippocampus and Prefrontal Cortex.

Adverse life experiences increase the lifetime risk to several stress-related psychopathologies, such as anxiety or depressive-like symptoms following stress in adulthood. However, the neurochemical modulations triggered by stress have not been fully characterized. Neuropeptides play an important role as signaling molecules that contribute to physiological regulation and have been linked to neurological and psychiatric diseases. However, little is known about the influence of stress on neuropeptide regulation in the brain. Here, we have performed an exploratory study of how neuropeptide expression at adulthood is modulated by experiencing a period of multiple stressful experiences. We have targeted hippocampus and prefrontal cortex (PFC) brain areas, which have previously been shown to be modulated by stressors, employing a targeted liquid chromatography-mass spectrometry (LC-MS) based approach that permits broad peptide coverage with high sensitivity. We found that in the hippocampus, Met-enkephalin, Met-enkephalin-Arg-Phe, and Met-enkephalin-Arg-Gly-Leu were upregulated, while Leu-enkephalin and Little SAAS were downregulated after stress. In the PFC area, Met-enkephalin-Arg-Phe, Met-enkephalin-Arg-Gly-Leu, peptide PHI-27, somatostatin-28 (AA1-12), and Little SAAS were all downregulated. This systematic evaluation of neuropeptide alterations in the hippocampus and PFC suggests that stressors impact neuropeptides and that neuropeptide regulation is brain-area specific. These findings suggest several potential peptide candidates, which warrant further investigations in terms of correlation with depression-associated behaviors.

Astrocytic and neuronal oxidative metabolism are coupled to the rate of glutamate-glutamine cycle in the tree shrew visual cortex.

Astrocytes play an important role in glutamatergic neurotransmission, namely by clearing synaptic glutamate and converting it into glutamine that is transferred back to neurons. The rate of this glutamate-glutamine cycle (VNT ) has been proposed to couple to that of glucose utilization and of neuronal tricarboxylic acid (TCA) cycle. In this study, we tested the hypothesis that glutamatergic neurotransmission is also coupled to the TCA cycle rate in astrocytes. For that we investigated energy metabolism by means of magnetic resonance spectroscopy (MRS) in the primary visual cortex of tree shrews (Tupaia belangeri) under light isoflurane anesthesia at rest and during continuous visual stimulation. After identifying the activated cortical volume by blood oxygenation level-dependent functional magnetic resonance imaging, 1 H MRS was performed to measure stimulation-induced variations in metabolite concentrations. Relative to baseline, stimulation of cortical activity for 20 min caused a reduction of glucose concentration by -0.34 ± 0.09 µmol/g (p < 0.001), as well as a -9% ± 1% decrease of the ratio of phosphocreatine-to-creatine (p < 0.05). Then 13 C MRS during [1,6-13 C]glucose infusion was employed to measure fluxes of energy metabolism. Stimulation of glutamatergic activity, as indicated by a 20% increase of VNT , resulted in increased TCA cycle rates in neurons by 12% ( VTCAn, p < 0.001) and in astrocytes by 24% ( VTCAg, p = 0.007). We further observed linear relationships between VNT and both VTCAn and VTCAg. Altogether, these results suggest that in the tree shrew primary visual cortex glutamatergic neurotransmission is linked to overall glucose oxidation and to mitochondrial metabolism in both neurons and astrocytes.

Separation and identification of mouse brain tissue microproteins using top-down method with high resolution nanocapillary liquid chromatography mass spectrometry.

Microproteins and endogenous peptides in the brain contain important substances that have critical roles in diverse biological processes, contributing to signal transduction and intercellular signaling. However, variability in their physical or chemical characteristics, such as molecule size, hydrophobicity, and charge states, complicate the simultaneous analysis of these compounds, although this would be highly beneficial for the field of neuroscience research. Here, we present a top-down analytical method for simultaneous analysis of microproteins and endogenous peptides using high-resolution nanocapillary LC-MS/MS. This method is detergent-free and digestion-free, which allows for extracting and preserving intact microproteins and peptides for direct LC-MS analysis. Both higher energy collision dissociation and electron-transfer dissociation fragmentations were used in the LC-MS analysis to increase the identification rate, and bioinformatics tools ProteinGoggle and PEAKS Studio software were utilized for database search. In total, we identified 471 microproteins containing 736 proteoforms, including brain-derived neurotrophic factor and a number of fibroblast growth factors. In addition, we identified 599 peptides containing 151 known or potential neuropeptides such as somatostatin-28 and neuropeptide Y. Our approach bridges the gap for the characterization of brain microproteins and peptides, which permits quantification of a diversity of signaling molecules for biomarker discovery or therapy diagnosis in the future.

Neural and neurochemical basis of reinforcement-guided decision making.

Decision making is an adaptive behavior that takes into account several internal and external input variables and leads to the choice of a course of action over other available and often competing alternatives. While it has been studied in diverse fields ranging from mathematics, economics, ecology, and ethology to psychology and neuroscience, recent cross talk among perspectives from different fields has yielded novel descriptions of decision processes. Reinforcement-guided decision making models are based on economic and reinforcement learning theories, and their focus is on the maximization of acquired benefit over a defined period of time. Studies based on reinforcement-guided decision making have implicated a large network of neural circuits across the brain. This network includes a wide range of cortical (e.g., orbitofrontal cortex and anterior cingulate cortex) and subcortical (e.g., nucleus accumbens and subthalamic nucleus) brain areas and uses several neurotransmitter systems (e.g., dopaminergic and serotonergic systems) to communicate and process decision-related information. This review discusses distinct as well as overlapping contributions of these networks and neurotransmitter systems to the processing of decision making. We end the review by touching on neural circuitry and neuromodulatory regulation of exploratory decision making.

Neural coding of image structure and contrast polarity of Cartesian, hyperbolic, and polar gratings in the primary and secondary visual cortex of the tree shrew.

We comprehensively characterize spiking and visual evoked potential (VEP) activity in tree shrew V1 and V2 using Cartesian, hyperbolic, and polar gratings. Neural selectivity to structure of Cartesian gratings was higher than other grating classes in both visual areas. From V1 to V2, structure selectivity of spiking activity increased, whereas corresponding VEP values tended to decrease, suggesting that single-neuron coding of Cartesian grating attributes improved while the cortical columnar organization of these neurons became less precise from V1 to V2. We observed that neurons in V2 generally exhibited similar selectivity for polar and Cartesian gratings, suggesting that structure of polar-like stimuli might be encoded as early as in V2. This hypothesis is supported by the preference shift from V1 to V2 toward polar gratings of higher spatial frequency, consistent with the notion that V2 neurons encode visual scene borders and contours. Neural sensitivity to modulations of polarity of hyperbolic gratings was highest among all grating classes and closely related to the visual receptive field (RF) organization of ON- and OFF-dominated subregions. We show that spatial RF reconstructions depend strongly on grating class, suggesting that intracortical contributions to RF structure are strongest for Cartesian and polar gratings. Hyperbolic gratings tend to recruit least cortical elaboration such that the RF maps are similar to those generated by sparse noise, which most closely approximate feedforward inputs. Our findings complement previous literature in primates, rodents, and carnivores and highlight novel aspects of shape representation and coding occurring in mammalian early visual cortex.

High-Efficiency Recognition and Identification of Disulfide Bonded Peptides in Rat Neuropeptidome Using Targeted Electron Transfer Dissociation Tandem Mass Spectrometry.

The main goal of the present study is to develop a method to recognize and identify endogenous intrachain disulfide bonded peptide, which are rarely sequenced in current peptidomics studies. In order to achieve highly efficient detection of these peptides in a neuropeptidome analysis, we alkylated the peptides, mined the raw mass spectrometry data, and then recognized the candidates of untreated disulfide bonded peptides from unalkylated peptide extracts. After removing more than 90% features, targeted electron transfer dissociation fragmentation was performed for detecting and fragmenting disulfide bonded peptides, and even most of them were present in low abundance in the original sample. Diverse endogenous disulfide bonded peptides were then detected and sequenced, opening up new perspectives for comprehensively understanding the response of a neuropeptidome.

On the relation between receptive field structure and stimulus selectivity in the tree shrew primary visual cortex.

There are notable differences in functional properties of primary visual cortex (V1) neurons among mammalian species, particularly those concerning the occurrence of simple and complex cells and the generation of orientation selectivity. Here, we present quantitative data on receptive field (RF) structure, response modulation, and orientation tuning for single neurons in V1 of the tree shrew, a close relative of primates. We find that spatial RF subfield segregation, a criterion for identifying simple cells, was exceedingly small in the tree shrew V1. In contrast, many neurons exhibited elevated F1/F0 modulation that is often used as a simple cell marker. This apparent discrepancy can be explained by the robust stimulus polarity preference in tree shrew V1, which inflates F1/F0 ratio values. RF structure mapped with sparse-noise-which is spatially restricted and emphasizes thalamo-cortical feed-forward inputs-appeared unrelated to orientation selectivity. However, RF structure mapped using the Hartley subspace stimulus-which covers a large area of the visual field and recruits considerable intracortical processing-did predict orientation preference. Our findings reveal a number of striking similarities in V1 functional organization between tree shrews and primates, emphasizing the important role of intracortical recurrent processing in shaping V1 response properties in these species.

Chronic nicotine treatment impacts the regulation of opioid and non-opioid peptides in the rat dorsal striatum.

The chronic use of nicotine, the main psychoactive ingredient of tobacco smoking, alters diverse physiological processes and consequently generates physical dependence. To understand the impact of chronic nicotine on neuropeptides, which are potential molecules associated with dependence, we conducted qualitative and quantitative neuropeptidomics on the rat dorsal striatum, an important brain region implicated in the preoccupation/craving phase of drug dependence. We used extensive LC-FT-MS/MS analyses for neuropeptide identification and LC-FT-MS in conjunction with stable isotope addition for relative quantification. The treatment with chronic nicotine for 3 months led to moderate changes in the levels of endogenous dorsal striatum peptides. Five enkephalin opioid peptides were up-regulated, although no change was observed for dynorphin peptides. Specially, nicotine altered levels of nine non-opioid peptides derived from precursors, including somatostatin and cerebellin, which potentially modulate neurotransmitter release and energy metabolism. This broad but selective impact on the multiple peptidergic systems suggests that apart from the opioid peptides, several other peptidergic systems are involved in the preoccupation/craving phase of drug dependence. Our finding permits future evaluation of the neurochemical circuits modulated by chronic nicotine exposure and provides a number of novel molecules that could serve as potential therapeutic targets for treating drug dependence.

Basal forebrain activation controls contrast sensitivity in primary visual cortex.

BACKGROUND: The basal forebrain (BF) regulates cortical activity by the action of cholinergic projections to the cortex. At the same time, it also sends substantial GABAergic projections to both cortex and thalamus, whose functional role has received far less attention. We used deep brain stimulation (DBS) in the BF, which is thought to activate both types of projections, to investigate the impact of BF activation on V1 neural activity. RESULTS: BF stimulation robustly increased V1 single and multi-unit activity, led to moderate decreases in orientation selectivity and a remarkable increase in contrast sensitivity as demonstrated by a reduced semi-saturation contrast. The spontaneous V1 local field potential often exhibited spectral peaks centered at 40 and 70 Hz as well as reliably showed a broad γ-band (30-90 Hz) increase following BF stimulation, whereas effects in a low frequency band (1-10 Hz) were less consistent. The broad γ-band, rather than low frequency activity or spectral peaks was the best predictor of both the firing rate increase and contrast sensitivity increase of V1 unit activity. CONCLUSIONS: We conclude that BF activation has a strong influence on contrast sensitivity in V1. We suggest that, in addition to cholinergic modulation, the BF GABAergic projections play a crucial role in the impact of BF DBS on cortical activity.

Deletion of the Circadian Clock Gene Per2 in the Whole Body, but Not in Neurons or Astroglia, Affects Sleep in Response to Sleep Deprivation.

The sleep-wake cycle is a highly regulated behavior in which a circadian clock times sleep and waking, whereas a homeostatic process controls sleep need. Both the clock and the sleep homeostat interact, but to what extent they influence each other is not understood. There is evidence that clock genes, in particular Period2 (Per2), might be implicated in the sleep homeostatic process. Sleep regulation depends also on the proper functioning of neurons and astroglial cells, two cell-types in the brain that are metabolically dependent on each other. In order to investigate clock-driven contributions to sleep regulation we non-invasively measured sleep of mice that lack the Per2 gene either in astroglia, neurons, or all body cells. We observed that mice lacking Per2 in all body cells (Per2Brdm and TPer2 animals) display earlier onset of sleep after sleep deprivation (SD), whereas neuronal and astroglial Per2 knock-out animals (NPer2 and GPer2, respectively) were normal in that respect. It appears that systemic (whole body) Per2 expression is important for physiological sleep architecture expressed by number and length of sleep bouts, whereas neuronal and astroglial Per2 weakly impacts night-time sleep amount. Our results suggest that Per2 contributes to the timing of the regulatory homeostatic sleep response by delaying sleep onset after SD and attenuating the early night rebound response.

Optogenetic activation of visual thalamus generates artificial visual percepts.

The lateral geniculate nucleus (LGN), a retinotopic relay center where visual inputs from the retina are processed and relayed to the visual cortex, has been proposed as a potential target for artificial vision. At present, it is unknown whether optogenetic LGN stimulation is sufficient to elicit behaviorally relevant percepts, and the properties of LGN neural responses relevant for artificial vision have not been thoroughly characterized. Here, we demonstrate that tree shrews pretrained on a visual detection task can detect optogenetic LGN activation using an AAV2-CamKIIα-ChR2 construct and readily generalize from visual to optogenetic detection. Simultaneous recordings of LGN spiking activity and primary visual cortex (V1) local field potentials (LFPs) during optogenetic LGN stimulation show that LGN neurons reliably follow optogenetic stimulation at frequencies up to 60 Hz and uncovered a striking phase locking between the V1 LFP and the evoked spiking activity in LGN. These phase relationships were maintained over a broad range of LGN stimulation frequencies, up to 80 Hz, with spike field coherence values favoring higher frequencies, indicating the ability to relay temporally precise information to V1 using light activation of the LGN. Finally, V1 LFP responses showed sensitivity values to LGN optogenetic activation that were similar to the animal's behavioral performance. Taken together, our findings confirm the LGN as a potential target for visual prosthetics in a highly visual mammal closely related to primates.

Dissociable effects of natural image structure and color on LFP and spiking activity in the lateral prefrontal cortex and extrastriate visual area V4.

Visual perception is mediated by unique contributions of the numerous brain regions that constitute the visual system. We performed simultaneous recordings of local field potentials (LFPs) and single unit activity (SUA) in areas V4 and lateral prefrontal cortex to characterize their contribution to visual processing. Here, we trained monkeys to identify natural images at different degradation levels in a visual recognition task. We parametrically varied color and structural information of natural images while the animals were performing the task. We show that the visual-evoked potential (VEP) of the LFP in V4 is highly sensitive to color, whereas the VEP in prefrontal cortex predominantly depends on image structure. When examining the relationship between VEP and SUA, we found that stimulus sensitivity for SUA was well predicted by the VEP in PF cortex but not in V4. Our results first reveal a functional specialization in both areas at the level of the LFP and further suggest that the degree to which mesoscopic signals, such as the VEP, are representative of the underlying SUA neural processing may be brain region specific within the context of visual recognition.

High identification rates of endogenous neuropeptides from mouse brain.

Mass spectrometry-based neuropeptidomics is one of the most powerful approaches for identification of endogenous neuropeptides in the brain. Until now, however, the identification rate of neuropeptides in neuropeptidomics is relatively low and this severely restricts insights into their biological function. In the present study, we developed a high accuracy mass spectrometry-based approach to enhance the identification rates of neuropeptides from brain tissue. Our integrated approach used mixing on column for loading aqueous and organic extracts to reduce the loss of peptides during sample treatment and used charge state-directed tandem mass spectrometry to increase the number of peptides subjected to high mass accuracy fragmentation. This approach allowed 206 peptides on average to be identified from a single mouse brain sample that was prepared using 15 µL of solutions per 1 mg of tissue. In total, we identified more than 500 endogenous peptides from mouse hypothalamus and whole brain samples. Our identification rate is about two to four times higher compared to previously reported studies conducted on mice or other species. The hydrophobic peptides, such as neuropeptide Y and galanin, could be presented and detected with hydrophilic peptides in the same LC-MS run, allowing a high coverage of peptide characterization over an organism. This will advance our understanding of the roles of diverse peptides and their links in the brain functions.

Extensive characterization of Tupaia belangeri neuropeptidome using an integrated mass spectrometric approach.

Neuropeptidomics is used to characterize endogenous peptides in the brain of tree shrews (Tupaia belangeri). Tree shrews are small animals similar to rodents in size but close relatives of primates, and are excellent models for brain research. Currently, tree shrews have no complete proteome information available on which direct database search can be allowed for neuropeptide identification. To increase the capability in the identification of neuropeptides in tree shrews, we developed an integrated mass spectrometry (MS)-based approach that combines methods including data-dependent, directed, and targeted liquid chromatography (LC)-Fourier transform (FT)-tandem MS (MS/MS) analysis, database construction, de novo sequencing, precursor protein search, and homology analysis. Using this integrated approach, we identified 107 endogenous peptides that have sequences identical or similar to those from other mammalian species. High accuracy MS and tandem MS information, with BLAST analysis and chromatographic characteristics were used to confirm the sequences of all the identified peptides. Interestingly, further sequence homology analysis demonstrated that tree shrew peptides have a significantly higher degree of homology to equivalent sequences in humans than those in mice or rats, consistent with the close phylogenetic relationship between tree shrews and primates. Our results provide the first extensive characterization of the peptidome in tree shrews, which now permits characterization of their function in nervous and endocrine system. As the approach developed fully used the conservative properties of neuropeptides in evolution and the advantage of high accuracy MS, it can be portable for identification of neuropeptides in other species for which the fully sequenced genomes or proteomes are not available.

Functional and laminar dissociations between muscarinic and nicotinic cholinergic neuromodulation in the tree shrew primary visual cortex.

Acetylcholine is an important neuromodulator involved in cognitive function. The impact of cholinergic neuromodulation on computations within the cortical microcircuit is not well understood. Here we investigate the effects of layer-specific cholinergic drug application in the tree shrew primary visual cortex during visual stimulation with drifting grating stimuli of varying contrast and orientation. We describe differences between muscarinic and nicotinic cholinergic effects in terms of both the layer of cortex and the attribute of visual representation. Nicotinic receptor activation enhanced the contrast response in the granular input layer of the cortex, while tending to reduce neural selectivity for orientation across all cortical layers. Muscarinic activation modestly enhanced the contrast response across cortical layers, and tended to improve orientation tuning. This resulted in highest orientation selectivity in the supra- and infragranular layers, where orientation selectivity was already greatest in the absence of pharmacological stimulation. Our results indicate that laminar position plays a crucial part in functional consequences of cholinergic stimulation, consistent with the differential distribution of cholinergic receptors. Nicotinic receptors function to enhance sensory representations arriving in the cortex, whereas muscarinic receptors act to boost the cortical computation of orientation tuning. Our findings suggest close homology between cholinergic mechanisms in tree shrew and primate visual cortices.

Overproduction of hydrogen sulfide, generated by cystathionine ß-synthase, disrupts brain wave patterns and contributes to neurobehavioral dysfunction in a rat model of down syndrome.

Using a novel rat model of Down syndrome (DS), the functional role of the cystathionine-ß-synthase (CBS)/hydrogen sulfide (H2S) pathway was investigated on the pathogenesis of brain wave pattern alterations and neurobehavioral dysfunction. Increased expression of CBS and subsequent overproduction of H2S was observed in the brain of DS rats, with CBS primarily localizing to astrocytes and the vasculature. DS rats exhibited neurobehavioral defects, accompanied by a loss of gamma brain wave activity and a suppression of the expression of multiple pre- and postsynaptic proteins. Aminooxyacetate, a prototypical pharmacological inhibitor of CBS, increased the ability of the DS brain tissue to generate ATP in vitro and reversed the electrophysiological and neurobehavioral alterations in vivo. Thus, the CBS/H2S pathway contributes to the pathogenesis of neurological dysfunction in DS, most likely through dysregulation of cellular bioenergetics and gene expression.

mTORC1 controls Golgi architecture and vesicle secretion by phosphorylation of SCYL1.

The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation, supporting anabolic reactions and inhibiting catabolic pathways like autophagy. Its hyperactivation is a frequent event in cancer promoting tumor cell proliferation. Several intracellular membrane-associated mTORC1 pools have been identified, linking its function to distinct subcellular localizations. Here, we characterize the N-terminal kinase-like protein SCYL1 as a Golgi-localized target through which mTORC1 controls organelle distribution and extracellular vesicle secretion in breast cancer cells. Under growth conditions, SCYL1 is phosphorylated by mTORC1 on Ser754, supporting Golgi localization. Upon mTORC1 inhibition, Ser754 dephosphorylation leads to SCYL1 displacement to endosomes. Peripheral, dephosphorylated SCYL1 causes Golgi enlargement, redistribution of early and late endosomes and increased extracellular vesicle release. Thus, the mTORC1-controlled phosphorylation status of SCYL1 is an important determinant regulating subcellular distribution and function of endolysosomal compartments. It may also explain the pathophysiology underlying human genetic diseases such as CALFAN syndrome, which is caused by loss-of-function of SCYL1.

Behavioral flexibility and the frontal lobe.

Brain areas in the frontal lobe have been implicated in behavioral flexibility and control. The study by Johnston et al. in this issue of Neuron provides novel insights into the roles of the prefrontal and anterior cingulate cortices in controlling behavior.