Sequential Activation of Lateral Hypothalamic Neuronal Populations during Feeding and Their Assembly by Gamma Oscillations.

Neural circuits supporting innate behaviors, such as feeding, exploration, and social interaction, intermingle in the lateral hypothalamus (LH). Although previous studies have shown that individual LH neurons change their firing relative to the baseline during one or more behaviors, the firing rate dynamics of LH populations within behavioral episodes and the coordination of behavior-related LH populations remain largely unknown. Here, using unsupervised graph-based clustering of LH neurons firing rate dynamics in freely behaving male mice, we identified distinct populations of cells whose activity corresponds to feeding, specific times during feeding bouts, or other innate behaviors-social interaction and novel object exploration. Feeding-related cells fired together with a higher probability during slow and fast gamma oscillations (30-60 and 60-90 Hz) than during nonrhythmic epochs. In contrast, the cofiring of neurons signaling other behaviors than feeding was overall similar between slow gamma and nonrhythmic epochs but increased during fast gamma oscillations. These results reveal a neural organization of ethological hierarchies in the LH and point to behavior-specific motivational systems, the dysfunction of which may contribute to mental disorders.

Mutations in plasticity-related-gene-1 (PRG-1) protein contribute to hippocampal seizure susceptibility and modify epileptic phenotype.

The Phospholipid Phosphatase Related 4 gene (PLPPR4,  *607813) encodes the Plasticity-Related-Gene-1 (PRG-1) protein. This cerebral synaptic transmembrane-protein modulates cortical excitatory transmission on glutamatergic neurons. In mice, homozygous Prg-1 deficiency causes juvenile epilepsy. Its epileptogenic potential in humans was unknown. Thus, we screened 18 patients with infantile epileptic spasms syndrome (IESS) and 98 patients with benign familial neonatal/infantile seizures (BFNS/BFIS) for the presence of PLPPR4 variants. A girl with IESS had inherited a PLPPR4-mutation (c.896C > G, NM_014839; p.T299S) from her father and an SCN1A-mutation from her mother (c.1622A > G, NM_006920; p.N541S). The PLPPR4-mutation was located in the third extracellular lysophosphatidic acid-interacting domain and in-utero electroporation (IUE) of the Prg-1p.T300S construct into neurons of Prg-1 knockout embryos demonstrated its inability to rescue the electrophysiological knockout phenotype. Electrophysiology on the recombinant SCN1Ap.N541S channel revealed partial loss-of-function. Another PLPPR4 variant (c.1034C > G, NM_014839; p.R345T) that was shown to result in a loss-of-function aggravated a BFNS/BFIS phenotype and also failed to suppress glutamatergic neurotransmission after IUE. The aggravating effect of Plppr4-haploinsufficiency on epileptogenesis was further verified using the kainate-model of epilepsy: double heterozygous Plppr4-/+|Scn1awt|p.R1648H mice exhibited higher seizure susceptibility than either wild-type, Plppr4-/+, or Scn1awt|p.R1648H littermates. Our study shows that a heterozygous PLPPR4 loss-of-function mutation may have a modifying effect on BFNS/BFIS and on SCN1A-related epilepsy in mice and humans.

Gamma oscillations organize top-down signalling to hypothalamus and enable food seeking.

Both humans and animals seek primary rewards in the environment, even when such rewards do not correspond to current physiological needs. An example of this is a dissociation between food-seeking behaviour and metabolic needs, a notoriously difficult-to-treat symptom of eating disorders. Feeding relies on distinct cell groups in the hypothalamus, the activity of which also changes in anticipation of feeding onset. The hypothalamus receives strong descending inputs from the lateral septum, which is connected, in turn, with cortical networks, but cognitive regulation of feeding-related behaviours is not yet understood. Cortical cognitive processing involves gamma oscillations, which support memory, attention, cognitive flexibility and sensory responses. These functions contribute crucially to feeding behaviour by unknown neural mechanisms. Here we show that coordinated gamma (30-90 Hz) oscillations in the lateral hypothalamus and upstream brain regions organize food-seeking behaviour in mice. Gamma-rhythmic input to the lateral hypothalamus from somatostatin-positive lateral septum cells evokes food approach without affecting food intake. Inhibitory inputs from the lateral septum enable separate signalling by lateral hypothalamus neurons according to their feeding-related activity, making them fire at distinct phases of the gamma oscillation. Upstream, medial prefrontal cortical projections provide gamma-rhythmic inputs to the lateral septum; these inputs are causally associated with improved performance in a food-rewarded learning task. Overall, our work identifies a top-down pathway that uses gamma synchronization to guide the activity of subcortical networks and to regulate feeding behaviour by dynamic reorganization of functional cell groups in the hypothalamus.

Microsatellite Instability in Russian Patients with Colorectal Cancer.

The aim of this study was to determine the characteristics of Russian patients with microsatellite instability (MSI) tumors. MSI in the tumor was determined in 514 patients with colon cancer using PCR and subsequent fragment analysis for five markers (NR21, NR24, BAT25, BAT26, and NR27). In the presence of microsatellite instability, the mismatch repair (MMR) system genes were examined using the NGS and MLPA methods to establish the diagnosis of Lynch syndrome. The overall frequency of MSI tumors was 15%: at stage I­19% (9/48), at stage II­21% (44/213), at stage III­16% (26/160), and at stage IV­2% (2/93). Patients with MSI tumors differed in the age of diagnosis, tumor localization, time of cancer recurrence, and stage of the disease. The overall and disease-free survival of patients whose tumors had MSI status was higher than that of patients with microsatellite-stable status, p = 0.04 and p = 0.02, respectively. Analysis of overall and disease-free survival of patients with Lynch syndrome and patients with sporadic colon cancer, but with MSI status, did not reveal significant differences, p = 0.52 and p = 0.24, respectively. The age of patients with Lynch syndrome was significantly younger than that of patients with sporadic colon cancer whose tumors had MSI status (p < 0.001).

Genetic and phenotypic characteristics of Russian patients with BRAF-mutated colorectal cancer.

Colorectal cancer (CRC) is one of the most common malignancies in the world. It's estimated about 1.8 M new CRC cases worldwide per year. A somatic mutation in the BRAF gene in the tumor is a negative prognostic factor. This work is aimed at studying the clinical and genetic characteristics of Russian CRC patients with the BRAF mutation. The BRAF mutations were studied by Sanger sequencing and digital droplet PCR in 489 patients and found in 34 (7%) cases. The most common mutation was p.V600E (82%). Also, rare variants were found: p.K601E, p.N581I, p.G596R, and p.D594N. All the patients with rare mutations were characterized by an unfavorable prognosis of the disease. The clinical features of the patients with BRAF mutations in the study include the predominant primary tumor site in the rectum, in addition to the right colon. Then, most of the cases were diagnosed in the advanced stages of the disease and were represented by high-grade adenocarcinomas. This article demonstrates the feasibility of analysis of the entire exon 15 of BRAF gene in CRC patients regardless of tumor localization.

Sleep & metabolism: The multitasking ability of lateral hypothalamic inhibitory circuitries.

The anatomical and functional mapping of lateral hypothalamic circuits has been limited by the numerous cell types and complex, yet unclear, connectivity. Recent advances in functional dissection of input-output neurons in the lateral hypothalamus have identified subset of inhibitory cells as crucial modulators of both sleep-wake states and metabolism. Here, we summarize these recent studies and discuss the multi-tasking functions of hypothalamic circuitries in integrating sleep and metabolism in the mammalian brain.

Syntaxin 1B is important for mouse postnatal survival and proper synaptic function at the mouse neuromuscular junctions.

STX1 is a major neuronal syntaxin protein located at the plasma membrane of the neuronal tissues. Rodent STX1 has two highly similar paralogs, STX1A and STX1B, that are thought to be functionally redundant. Interestingly, some studies have shown that the distribution patterns of STX1A and STX1B at the central and peripheral nervous systems only partially overlapped, implying that there might be differential functions between these paralogs. In the current study, we generated an STX1B knockout (KO) mouse line and studied the impact of STX1B removal in neurons of several brain regions and the neuromuscular junction (NMJ). We found that either complete removal of STX1B or selective removal of it from forebrain excitatory neurons in mice caused premature death. Autaptic hippocampal and striatal cultures derived from STX1B KO mice still maintained efficient neurotransmission compared with neurons from STX1B wild-type and heterozygous mice. Interestingly, examining high-density cerebellar cultures revealed a decrease in the spontaneous GABAergic transmission frequency, which was most likely due to a lower number of neurons in the STX1B KO cultures, suggesting that STX1B is essential for neuronal survival in vitro. Moreover, our study also demonstrated that although STX1B is dispensable for the formation of the mouse NMJ, it is required to maintain the efficiency of neurotransmission at the nerve-muscle synapse.

The dynamic state of a prefrontal-hypothalamic-midbrain circuit commands behavioral transitions.

Innate behaviors meet multiple needs adaptively and in a serial order, suggesting the existence of a hitherto elusive brain dynamics that brings together representations of upcoming behaviors during their selection. Here we show that during behavioral transitions, possible upcoming behaviors are encoded by specific signatures of neuronal populations in the lateral hypothalamus (LH) that are active near beta oscillation peaks. Optogenetic recruitment of intrahypothalamic inhibition at this phase eliminates behavioral transitions. We show that transitions are elicited by beta-rhythmic inputs from the prefrontal cortex that spontaneously synchronize with LH 'transition cells' encoding multiple behaviors. Downstream of the LH, dopamine neurons increase firing during beta oscillations and also encode behavioral transitions. Thus, a hypothalamic transition state signals alternative future behaviors, encodes the one most likely to be selected and enables rapid coordination with cognitive and reward-processing circuitries, commanding adaptive social contact and eating behaviors.

Cannabinoids disrupt hippocampal sharp wave-ripples via inhibition of glutamate release.

Cannabis consumption results in impaired learning. The proper synchronization of neuronal activity in the mammalian hippocampus gives rise to network rhythms that are implicated in memory formation. Here, we have studied the impact of cannabinoids on hippocampal sharp waves and associated ripple oscillations using field- and whole-cell voltage-clamp recordings. We demonstrate that the activation of cannabinoid receptor 1 suppresses sharp wave-ripples (SWRs) in mice in vivo and in vitro. This suppression was paralleled by a selective reduction of SWR-associated inward but not outward charge transfer, demonstrating an impairment of excitation due to cannabinoid exposure. Adenosine, a presynaptic modulator of glutamate release, mimicked and occluded the observed consequences of cannabinoids on SWRs. We conclude that inhibition of glutamatergic feed-forward excitation can explain cannabinoid-mediated disruption of SWRs and may account for cannabinoid-induced impairment of hippocampus-dependent memory.

Hippocampal theta rhythm and its coupling with gamma oscillations require fast inhibition onto parvalbumin-positive interneurons.

Hippocampal theta (5-10 Hz) and gamma (35-85 Hz) oscillations depend on an inhibitory network of GABAergic interneurons. However, the lack of methods for direct and cell-type-specific interference with inhibition has prevented better insights that help link synaptic and cellular properties with network function. Here, we generated genetically modified mice (PV-Deltagamma(2)) in which synaptic inhibition was ablated in parvalbumin-positive (PV+) interneurons. Hippocampal local field potential and unit recordings in the CA1 area of freely behaving mice revealed that theta rhythm was strongly reduced in these mice. The characteristic coupling of theta and gamma oscillations was strongly altered in PV-Deltagamma(2) mice more than could be accounted for by the reduction in theta rhythm only. Surprisingly, gamma oscillations were not altered. These data indicate that synaptic inhibition onto PV+ interneurons is indispensable for theta- and its coupling to gamma oscillations but not for rhythmic gamma-activity in the hippocampus. Similar alterations in rhythmic activity were obtained in a computational hippocampal network model mimicking the genetic modification, suggesting that intrahippocampal networks might contribute to these effects.

GABAergic signaling to astrocytes in the prefrontal cortex sustains goal-directed behaviors.

GABA interneurons play a critical role in higher brain functions. Astrocytic glial cells interact with synapses throughout the whole brain and are recognized as regulatory elements of excitatory synaptic transmission. However, it is largely unknown how GABAergic interneurons and astrocytes interact and contribute to stable performance of complex behaviors. Here, we found that genetic ablation of GABAB receptors in medial prefrontal cortex astrocytes altered low-gamma oscillations and firing properties of cortical neurons, which affected goal-directed behaviors. Remarkably, working memory deficits were restored by optogenetic stimulation of astrocytes with melanopsin. Furthermore, melanopsin-activated astrocytes in wild-type mice enhanced the firing rate of cortical neurons and gamma oscillations, as well as improved cognition. Therefore, our work identifies astrocytes as a hub for controlling inhibition in cortical circuits, providing a novel pathway for the behaviorally relevant midrange time-scale regulation of cortical information processing and consistent goal-directed behaviors.

Place fields of single spikes in hippocampus involve Kcnq3 channel-dependent entrainment of complex spike bursts.

Hippocampal pyramidal cells encode an animal's location by single action potentials and complex spike bursts. These elementary signals are believed to play distinct roles in memory consolidation. The timing of single spikes and bursts is determined by intrinsic excitability and theta oscillations (5-10 Hz). Yet contributions of these dynamics to place fields remain elusive due to the lack of methods for specific modification of burst discharge. In mice lacking Kcnq3-containing M-type K+ channels, we find that pyramidal cell bursts are less coordinated by the theta rhythm than in controls during spatial navigation, but not alert immobility. Less modulated bursts are followed by an intact post-burst pause of single spike firing, resulting in a temporal discoordination of network oscillatory and intrinsic excitability. Place fields of single spikes in one- and two-dimensional environments are smaller in the mutant. Optogenetic manipulations of upstream signals reveal that neither medial septal GABA-ergic nor cholinergic inputs alone, but rather their joint activity, is required for entrainment of bursts. Our results suggest that altered representations by bursts and single spikes may contribute to deficits underlying cognitive disabilities associated with KCNQ3-mutations in humans.

Augmented hippocampal ripple oscillations in mice with reduced fast excitation onto parvalbumin-positive cells.

Generation of fast network oscillations in the hippocampus relies on interneurons, but the underlying specific synaptic mechanisms are not established. The excitatory recruitment of fast-spiking interneurons during hippocampal sharp waves has been suggested to be critical for the generation of 140-200 Hz ("ripple") oscillations in the CA1 area. To directly test this, we used genetically modified mice (PV-DeltaGluR-A) with reduced AMPA receptor-mediated excitation onto parvalbumin (PV)-positive interneurons and studied hippocampal oscillations in freely moving animals. In PV-DeltaGluR-A mice, ripple-amplitude and associated rhythmic modulation of pyramidal cells and fast-spiking interneurons were increased. These changes were not accompanied by concurrent alterations of firing rates. Neither theta nor gamma oscillations displayed marked alterations in the mutant. These results provide evidence that fast excitation from pyramidal cells to PV-positive interneurons differentially influences ripple and gamma oscillations in vivo.

Prefrontal - subthalamic pathway supports action selection in a spatial working memory task.

Subthalamic nucleus (STN) is the main source of feed-forward excitation in the basal ganglia and a main target of therapeutic deep brain stimulation in movement disorders. Alleviation of motor symptoms during STN stimulation can be accompanied by deterioration of abilities to quickly choose between conflicting alternatives. Cortical afferents to the subthalamic region (ST), comprising STN and zona incerta (ZI), include projections from the medial prefrontal cortex (mPFC), yet little is known about prefrontal-subthalamic coordination and its relevance for decision-making. Here we combined electrophysiological recordings with optogenetic manipulations of projections from mPFC to ST in mice as they performed a spatial working memory task (T-maze) or explored an elevated plus maze (anxiety test). We found that gamma oscillations (30-70 Hz) are coordinated between mPFC and ST at theta (5-10 Hz) and, less efficiently, at sub-theta (2-5 Hz) frequencies. An optogenetic detuning of the theta/gamma cross-frequency coupling between the regions into sub-theta range impaired performance in the T-maze, yet did not affect anxiety-related behaviors in the elevated plus maze. Both detuning and inhibition of the mPFC-ST pathway led to repeated incorrect choices in the T-maze. These effects were not associated with changes of anxiety and motor activity measures. Our findings suggest that action selection in a cognitively demanding task crucially involves theta rhythmic coordination of gamma oscillatory signaling in the prefrontal-subthalamic pathway.

Frequency of network synchronization in the hippocampus marks learning.

The synchronization of neuronal networks may be instrumental in plasticity and learning. Hippocampal high-frequency oscillations (140-200 Hz, 'ripples') characteristic of consummatory behaviours are thought to promote memory formation. We recorded ripple oscillations from the CA1 area in temporal learning tasks. Rats learned to adjust their operant response to the timing of food reward delivery [fixed interval schedule (FI)]. The intrinsic frequency of ripples was elevated following the switch in reinforcement timing. Learning, as assessed from the response pattern, correlated with fluctuations of intraripple frequency and amplitude. Changes in motor activity did not account for the variability of ripple oscillations. At the same time, features of ripples were unaltered when the fixed interval of reward delivery was changed but did not depend on the lever press response. Thus, in addition to the known replay of neuronal firing patterns during ripple oscillations, the rhythm itself appears to be modulated in an experience-specific way and represents a direct correlate of learning.

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice.

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

Reconciling the different faces of hippocampal theta: The role of theta oscillations in cognitive, emotional and innate behaviors.

The theta oscillation (5-10Hz) is a prominent behavior-specific brain rhythm. This review summarizes studies showing the multifaceted role of theta rhythm in cognitive functions, including spatial coding, time coding and memory, exploratory locomotion and anxiety-related behaviors. We describe how activity of hippocampal theta rhythm generators - medial septum, nucleus incertus and entorhinal cortex, links theta with specific behaviors. We review evidence for functions of the theta-rhythmic signaling to subcortical targets, including lateral septum. Further, we describe functional associations of theta oscillation properties - phase, frequency and amplitude - with memory, locomotion and anxiety, and outline how manipulations of these features, using optogenetics or pharmacology, affect associative and innate behaviors. We discuss work linking cognition to the slope of the theta frequency to running speed regression, and emotion-sensitivity (anxiolysis) to its y-intercept. Finally, we describe parallel emergence of theta oscillations, theta-mediated neuronal activity and behaviors during development. This review highlights a complex interplay of neuronal circuits and synchronization features, which enables an adaptive regulation of multiple behaviors by theta-rhythmic signaling.

Hypothalamic feedforward inhibition of thalamocortical network controls arousal and consciousness.

During non-rapid eye movement (NREM) sleep, synchronous synaptic activity in the thalamocortical network generates predominantly low-frequency oscillations (<4 Hz) that are modulated by inhibitory inputs from the thalamic reticular nucleus (TRN). Whether TRN cells integrate sleep-wake signals from subcortical circuits remains unclear. We found that GABA neurons from the lateral hypothalamus (LHGABA) exert a strong inhibitory control over TRN GABA neurons (TRNGABA). We found that optogenetic activation of this circuit recapitulated state-dependent changes of TRN neuron activity in behaving mice and induced rapid arousal during NREM, but not REM, sleep. During deep anesthesia, activation of this circuit induced sustained cortical arousal. In contrast, optogenetic silencing of LHGABA-TRNGABA transmission increased the duration of NREM sleep and amplitude of delta (1-4 Hz) oscillations. Collectively, these results demonstrate that TRN cells integrate subcortical arousal inputs selectively during NREM sleep and may participate in sleep intensity.

Cannabinoid Type 2 Receptors Mediate a Cell Type-Specific Plasticity in the Hippocampus.

Endocannabinoids (eCBs) exert major control over neuronal activity by activating cannabinoid receptors (CBRs). The functionality of the eCB system is primarily ascribed to the well-documented retrograde activation of presynaptic CB1Rs. We find that action potential-driven eCB release leads to a long-lasting membrane potential hyperpolarization in hippocampal principal cells that is independent of CB1Rs. The hyperpolarization, which is specific to CA3 and CA2 pyramidal cells (PCs), depends on the activation of neuronal CB2Rs, as shown by a combined pharmacogenetic and immunohistochemical approach. Upon activation, they modulate the activity of the sodium-bicarbonate co-transporter, leading to a hyperpolarization of the neuron. CB2R activation occurred in a purely self-regulatory manner, robustly altered the input/output function of CA3 PCs, and modulated gamma oscillations in vivo. To conclude, we describe a cell type-specific plasticity mechanism in the hippocampus that provides evidence for the neuronal expression of CB2Rs and emphasizes their importance in basic neuronal transmission.