Olfactory bulb drives respiration-coupled beta oscillations in the rat hippocampus.

The synchronization of neuronal oscillations has been suggested as a mechanism to coordinate information flow between distant brain regions. In particular, the olfactory bulb (OB) and the hippocampus (HPC) have been shown to exhibit oscillations in the beta frequency range (10-20 Hz) that are likely to support communication between these structures. Here, we further characterize features of beta oscillations in OB and HPC of rats anesthetized with urethane. We find that beta oscillations simultaneously appear in HPC and OB and phase-lock across structures. Moreover, Granger causality analysis reveals that OB beta activity drives HPC beta. The laminar voltage profile of beta in HPC shows the maximum amplitude in the dentate gyrus (DG), spatially coinciding with olfactory inputs to this region. Finally, we also find that the respiratory cycle and respiration-coupled field potential rhythms (1-2 Hz)-but not theta oscillations (3-5 Hz)-modulate beta amplitude in OB and HPC. In all, our results support the hypothesis that beta activity mediates the communication between olfactory and hippocampal circuits in the rodent brain.

Rats Synchronize Locomotion with Ultrasonic Vocalizations at the Subsecond Time Scale.

Acoustic signals have the potential for transmitting information fast across distances. Rats emit ultrasonic vocalizations of two distinct classes: "22-kHz" or "alarm" calls and "50-kHz" calls. The latter comprises brief sounds in the 30-80-kHz range, whose ethological role is not fully understood. We recorded ultrasonic vocalizations from pairs of rats freely behaving in neighboring but separated arenas. 50-kHz vocalizations in this condition were tightly linked to the locomotion of the emitter at the subsecond time scale, their rate sharply increasing and decreasing prior to the onset and offset of movement respectively. This locomotion-linked vocalization behavior showed a clear "audience effect," as rats recorded alone displayed lower vocal production than rats in social settings for equivalent speeds of locomotion. Furthermore, calls from different categories across the 50 and 22-kHz families displayed markedly different correlations with locomotor activity. Our results show that rat vocalizations in the high ultrasonic range are social signals carrying spatial information about the emitter and highlight the possibility that they may play a role in the social coordination of spatial behaviors.

A Respiration-Coupled Rhythm in the Rat Hippocampus Independent of Theta and Slow Oscillations.

UNLABELLED: During slow-wave sleep and deep anesthesia, the rat hippocampus displays a slow oscillation (SO) that follows "up-and-down" state transitions in the neocortex. There has been recent debate as to whether this local field potential (LFP) rhythm reflects internal processing or entrains with respiratory inputs. To solve this issue, here we have concomitantly recorded respiration along with hippocampal, neocortical, and olfactory bulb (OB) LFPs in rats anesthetized with urethane. During the course of anesthesia, LFPs transitioned between activity states characterized by the emergence of different oscillations. By jointly analyzing multisite LFPs and respiratory cycles, we could distinguish three types of low-frequency hippocampal oscillations: (1) SO, which coupled to neocortical up-and-down transitions; (2) theta, which phase-reversed across hippocampal layers and was largest at the fissure; and (3) a low-frequency rhythm with largest amplitude in the dentate gyrus, which coupled to respiration-entrained oscillations in OB and to respiration itself. In contrast, neither theta nor SO coupled to respiration. The hippocampal respiration-coupled rhythm and SO had frequency <1.5 Hz, whereas theta tended to be faster (>3 Hz). Tracheotomy abolished hippocampal respiration-coupled rhythm, which was restored by rhythmic delivery of air puffs into the nasal cavity. These results solve the apparent contradictions among previous studies by demonstrating that the rat hippocampus produces multiple types of low-frequency oscillations. Because they synchronize with different brain circuits, however, we postulate that each activity pattern plays a unique role in information processing. SIGNIFICANCE STATEMENT: The rat hippocampus exhibits a large-amplitude slow oscillation (<1.5 Hz) during deep sleep and anesthesia. It is currently debated whether this rhythm reflects internal processing with the neocortex or entrainment by external inputs from rhythmic nasal respiration, which has similar frequency. Here we reconcile previous studies by showing that the hippocampus can actually produce two low-frequency rhythms at nearby frequencies: one that indeed couples to respiration and another that is coupled to the neocortex. We further show that the respiration-coupled rhythm differs from theta oscillations. The results support a role for brain oscillations in connecting distant brain regions, and posit the respiratory cycle as an important reference for neuronal communication between olfactory and memory networks.

Emergence of invariant representation of vocalizations in the auditory cortex.

An essential task of the auditory system is to discriminate between different communication signals, such as vocalizations. In everyday acoustic environments, the auditory system needs to be capable of performing the discrimination under different acoustic distortions of vocalizations. To achieve this, the auditory system is thought to build a representation of vocalizations that is invariant to their basic acoustic transformations. The mechanism by which neuronal populations create such an invariant representation within the auditory cortex is only beginning to be understood. We recorded the responses of populations of neurons in the primary and nonprimary auditory cortex of rats to original and acoustically distorted vocalizations. We found that populations of neurons in the nonprimary auditory cortex exhibited greater invariance in encoding vocalizations over acoustic transformations than neuronal populations in the primary auditory cortex. These findings are consistent with the hypothesis that invariant representations are created gradually through hierarchical transformation within the auditory pathway.

Rodent ultrasonic vocalizations are bound to active sniffing behavior.

During rodent active behavior, multiple orofacial sensorimotor behaviors, including sniffing and whisking, display rhythmicity in the theta range (~5-10 Hz). During specific behaviors, these rhythmic patterns interlock, such that execution of individual motor programs becomes dependent on the state of the others. Here we performed simultaneous recordings of the respiratory cycle and ultrasonic vocalization emission by adult rats and mice in social settings. We used automated analysis to examine the relationship between breathing patterns and vocalization over long time periods. Rat ultrasonic vocalizations (USVs, "50 kHz") were emitted within stretches of active sniffing (5-10 Hz) and were largely absent during periods of passive breathing (1-4 Hz). Because ultrasound was tightly linked to the exhalation phase, the sniffing cycle segmented vocal production into discrete calls and imposed its theta rhythmicity on their timing. In turn, calls briefly prolonged exhalations, causing an immediate drop in sniffing rate. Similar results were obtained in mice. Our results show that ultrasonic vocalizations are an integral part of the rhythmic orofacial behavioral ensemble. This complex behavioral program is thus involved not only in active sensing but also in the temporal structuring of social communication signals. Many other social signals of mammals, including monkey calls and human speech, show structure in the theta range. Our work points to a mechanism for such structuring in rodent ultrasonic vocalizations.

Cross-modal responses in the primary visual cortex encode complex objects and correlate with tactile discrimination.

Cortical areas that directly receive sensory inputs from the thalamus were long thought to be exclusively dedicated to a single modality, originating separate labeled lines. In the past decade, however, several independent lines of research have demonstrated cross-modal responses in primary sensory areas. To investigate whether these responses represent behaviorally relevant information, we carried out neuronal recordings in the primary somatosensory cortex (S1) and primary visual cortex (V1) of rats as they performed whisker-based tasks in the dark. During the free exploration of novel objects, V1 and S1 responses carried comparable amounts of information about object identity. During execution of an aperture tactile discrimination task, tactile recruitment was slower and less robust in V1 than in S1. However, V1 tactile responses correlated significantly with performance across sessions. Altogether, the results support the notion that primary sensory areas have a preference for a given modality but can engage in meaningful cross-modal processing depending on task demand.

Neurons born in the adult dentate gyrus form functional synapses with target cells.

Adult neurogenesis occurs in the hippocampus and the olfactory bulb of the mammalian CNS. Recent studies have demonstrated that newborn granule cells of the adult hippocampus are postsynaptic targets of excitatory and inhibitory neurons, but evidence of synapse formation by the axons of these cells is still lacking. By combining retroviral expression of green fluorescent protein in adult-born neurons of the mouse dentate gyrus with immuno-electron microscopy, we found output synapses that were formed by labeled terminals on appropriate target cells in the CA3 area and the hilus. Furthermore, retroviral expression of channelrhodopsin-2 allowed us to light-stimulate newborn granule cells and identify postsynaptic target neurons by whole-cell recordings in acute slices. Our structural and functional evidence indicates that axons of adult-born granule cells establish synapses with hilar interneurons, mossy cells and CA3 pyramidal cells and release glutamate as their main neurotransmitter.

A DNA vaccine coding for the chimera BLSOmp31 induced a better degree of protection against B. ovis and a similar degree of protection against B. melitensis than Rev.1 vaccination.

In the present study, we reported an attempt to improve the immunogenicity and protective capacity of the chimera BLSOmp31 using a different antigen delivery: DNA vaccination. Vaccination of BALB/c mice with the DNA vaccine coding for the chimera BLSOmp31 (pCIBLSOmp31) provided the best protection level against Brucella ovis, which was significantly higher than the given by the co-delivery of both plasmids coding for the whole proteins (pcDNABLS+pCIOmp31) and even higher than the control vaccine Rev.1. Moreover, pCIBLSOmp31 induced higher protection against Brucella melitensis than pcDNABLS+pCIOmp31 but similar protection than Rev.1. The chimera induced a strong humoral response against the inserted peptide. It also induced peptide- and BLS-specific cytotoxic T responses. The insertion of this peptide on BLS induced stronger T helper 1 responses specific for the carrier (BLS), thus our results represent a case of synergic strengthening between two Brucella antigens. Hitherto, this is the first indication that a recombinant subunit vaccine elicits greater protection than whole Brucella.

Similar GABAergic inputs in dentate granule cells born during embryonic and adult neurogenesis.

Neurogenesis in the dentate gyrus of the hippocampus follows a unique temporal pattern that begins during embryonic development, peaks during the early postnatal stages and persists through adult life. We have recently shown that dentate granule cells born in early postnatal and adult mice acquire a remarkably similar afferent connectivity and firing behavior, suggesting that they constitute a homogeneous functional population [Laplagne et al. (2006)PLoS Biol., 4, e409]. Here we extend our previous study by comparing mature neurons born in the embryonic and adult hippocampus, with a focus on intrinsic membrane properties and gamma-aminobutyric acid (GABA)ergic synaptic inputs. For this purpose, dividing neuroblasts of the ventricular wall were retrovirally labeled with green fluorescent protein at embryonic day 15 (E15), and progenitor cells of the subgranular zone were labeled with red fluorescent protein in the same mice at postnatal day 42 (P42, adulthood). Electrophysiological properties of mature neurons born at either stage were then compared in the same brain slices. Evoked and spontaneous GABAergic postsynaptic responses of perisomatic and dendritic origin displayed similar characteristics in both neuronal populations. Miniature GABAergic inputs also showed similar functional properties and pharmacological profile. A comparative analysis of the present data with our previous observations rendered no significant differences among GABAergic inputs recorded from neurons born in the embryonic, early postnatal and adult mice. Yet, embryo-born neurons showed a reduced membrane excitability, suggesting a lower engagement in network activity. Our results demonstrate that granule cells of different age, location and degree of excitability receive GABAergic inputs of equivalent functional characteristics.

A recombinant subunit vaccine based on the insertion of 27 amino acids from Omp31 to the N-terminus of BLS induced a similar degree of protection against B. ovis than Rev.1 vaccination.

The development of an effective subunit vaccine against brucellosis is a research area of intense interest. The enzyme lumazine synthase from Brucella spp. (BLS) is highly immunogenic, presumably due to its decameric arrangement and remarkable stability. In this work we decided to develop a chimera with the scaffold protein BLS decorated with 10 copies of a known protective epitope derived from an outer membrane protein of 31kDa (Omp31) from Brucella spp. Vaccination of BALB/c mice with the chimera as a recombinant protein (rBLSOmp31) provided the best protection level against Brucella ovis, which was higher than the given by the co-delivery of both recombinant proteins (rBLS + rOmp31) and similar than the control vaccine Brucella melitensis strain Rev.1. Moreover rBLSOmp31 induced protection against Brucella melitensis but to a lesser degree than Rev.1. The chimera induced a strong humoral response against the inserted peptide. It also induced peptide- and BLS-specific T helper 1 and cytotoxic T responses. In conclusion, our results indicate that BLSOmp31 could be a useful candidate for the development of subunit vaccines against brucellosis since it elicits humoral, T helper and cytotoxic immune responses and protection against smooth and rough species of Brucella.

Functional convergence of neurons generated in the developing and adult hippocampus.

The dentate gyrus of the hippocampus contains neural progenitor cells (NPCs) that generate neurons throughout life. Developing neurons of the adult hippocampus have been described in depth. However, little is known about their functional properties as they become fully mature dentate granule cells (DGCs). To compare mature DGCs generated during development and adulthood, NPCs were labeled at both time points using retroviruses expressing different fluorescent proteins. Sequential electrophysiological recordings from neighboring neurons of different ages were carried out to quantitatively study their major synaptic inputs: excitatory projections from the entorhinal cortex and inhibitory afferents from local interneurons. Our results show that DGCs generated in the developing and adult hippocampus display a remarkably similar afferent connectivity with regard to both glutamate and GABA, the major neurotransmitters. We also demonstrate that adult-born neurons can fire action potentials in response to an excitatory drive, exhibiting a firing behavior comparable to that of neurons generated during development. We propose that neurons born in the developing and adult hippocampus constitute a functionally homogeneous neuronal population. These observations are critical to understanding the role of adult neurogenesis in hippocampal function.

Neuronal differentiation in the adult hippocampus recapitulates embryonic development.

In the adult hippocampus and olfactory bulb, neural progenitor cells generate neurons that functionally integrate into the existing circuits. To understand how neuronal differentiation occurs in the adult hippocampus, we labeled dividing progenitor cells with a retrovirus expressing green fluorescent protein and studied the morphological and functional properties of their neuronal progeny over the following weeks. During the first week neurons had an irregular shape and immature spikes and were synaptically silent. Slow GABAergic synaptic inputs first appeared during the second week, when neurons exhibited spineless dendrites and migrated into the granule cell layer. In contrast, glutamatergic afferents were detected by the fourth week in neurons displaying mature excitability and morphology. Interestingly, fast GABAergic responses were the latest to appear. It is striking that neuronal maturation in the adult hippocampus follows a precise sequence of connectivity (silent --> slow GABA --> glutamate --> fast GABA) that resembles hippocampal development. We conclude that, unlike what is observed in the olfactory bulb, the hippocampus maintains the same developmental rules for neuronal integration through adulthood.

Sequence determinants of quaternary structure in lumazine synthase.

Riboflavin, an essential cofactor for all organisms, is biosynthesized in plants, fungi and microorganisms. The penultimate step in the pathway is catalyzed by the enzyme lumazine synthase. One of the most distinctive characteristics of this enzyme is that it is found in different species in two different quaternary structures, pentameric and icosahedral, built from practically the same structural monomeric unit. In fact, the icosahedral structure is best described as a capsid of twelve pentamers. Despite this noticeable difference, the active sites are virtually identical in all structurally studied members. Furthermore, the main regions involved in the catalysis are located at the interface between adjacent subunits in the pentamer. Thus, the two quaternary forms of the enzyme must meet similar structural requirements to achieve their function, but, at the same time, they should differ in the sequence traits responsible for the different quaternary structures observed. Here, we present a combined analysis that includes sequence-structure and evolutionary studies to find the sequence determinants of the different quaternary assemblies of this enzyme. A data set containing 86 sequences of the lumazine synthase family was recovered by sequence similarity searches. Seven of them had resolved three-dimensional structures. A subsequent phylogenetic reconstruction by maximum parsimony (MP) allowed division of the total set into two clusters in accord with their quaternary structure. The comparison between the patterns of three-dimensional contacts derived from the known three-dimensional structures and variation in sequence conservation revealed a significant shift in structural constraints of certain positions. Also, to explore the changes in functional constraints between the two groups, site-specific evolutionary rate shifts were analyzed. We found that the positions involved in icosahedral contacts suffer a larger increase in constraints than the rest. We found eight sequence sites that would be the most important icosahedral sequence determinants. We discuss our results and compare them with previous work. These findings should contribute to refinement of the current structural data, to the design of assays that explore the role of these positions, to the structural characterization of new sequences, and to initiation of a study of the underlying evolutionary mechanisms.