Neuronal Response Latencies Encode First Odor Identity Information across Subjects.

Odorants are coded in the primary olfactory processing centers by spatially and temporally distributed patterns of glomerular activity. Whereas the spatial distribution of odorant-induced responses is known to be conserved across individuals, the universality of its temporal structure is still debated. Via fast two-photon calcium imaging, we analyzed the early phase of neuronal responses in the form of the activity onset latencies in the antennal lobe projection neurons of honeybee foragers. We show that each odorant evokes a stimulus-specific response latency pattern across the glomerular coding space. Moreover, we investigate these early response features for the first time across animals, revealing that the order of glomerular firing onsets is conserved across individuals and allows them to reliably predict odorant identity, but not concentration. These results suggest that the neuronal response latencies provide the first available code for fast odor identification.SIGNIFICANCE STATEMENT Here, we studied early temporal coding in the primary olfactory processing centers of the honeybee brain by fast imaging of glomerular responses to different odorants across glomeruli and across individuals. Regarding the elusive role of rapid response dynamics in olfactory coding, we were able to clarify the following aspects: (1) the rank of glomerular activation is conserved across individuals, (2) its stimulus prediction accuracy is equal to that of the response amplitude code, and (3) it contains complementary information. Our findings suggest a substantial role of response latencies in odor identification, anticipating the static response amplitude code.

In the Piriform Cortex, the Primary Impetus for Information Encoding through Synaptic Plasticity Is Provided by Descending Rather than Ascending Olfactory Inputs.

Information encoding by means of persistent changes in synaptic strength supports long-term information storage and memory in structures such as the hippocampus. In the piriform cortex (PC), that engages in the processing of associative memory, only short-term synaptic plasticity has been described to date, both in vitro and in anesthetized rodents in vivo. Whether the PC maintains changes in synaptic strength for longer periods of time is unknown: Such a property would indicate that it can serve as a repository for long-term memories. Here, we report that in freely behaving animals, frequency-dependent synaptic plasticity does not occur in the anterior PC (aPC) following patterned stimulation of the olfactory bulb (OB). Naris closure changed action potential properties of aPC neurons and enabled expression of long-term potentiation (LTP) by OB stimulation, indicating that an intrinsic ability to express synaptic plasticity is present. Odor discrimination and categorization in the aPC is supported by descending inputs from the orbitofrontal cortex (OFC). Here, OFC stimulation resulted in LTP (>4 h), suggesting that this structure plays an important role in promoting information encoding through synaptic plasticity in the aPC. These persistent changes in synaptic strength are likely to comprise a means through which long-term memories are encoded and/or retained in the PC.

Circuit formation and function in the olfactory bulb of mice with reduced spontaneous afferent activity.

The type of neuronal activity required for circuit development is a matter of significant debate. We addressed this issue by analyzing the topographic organization of the olfactory bulb in transgenic mice engineered to have very little afferent spontaneous activity due to the overexpression of the inwardly rectifying potassium channel Kir2.1 in the olfactory sensory neurons (Kir2.1 mice). In these conditions, the topography of the olfactory bulb was unrefined. Odor-evoked responses were readily recorded in glomeruli with reduced spontaneous afferent activity, although the functional maps were coarser than in controls and contributed to altered olfactory discrimination behavior. In addition, overexpression of Kir2.1 in adults induced a regression of the already refined connectivity to an immature (i.e., coarser) status. Our data suggest that spontaneous activity plays a critical role not only in the development but also in the maintenance of the topography of the olfactory bulb and in sensory information processing.

Illuminating vertebrate olfactory processing.

The olfactory system encodes information about molecules by spatiotemporal patterns of activity across distributed populations of neurons and extracts information from these patterns to control specific behaviors. Recent studies used in vivo recordings, optogenetics, and other methods to analyze the mechanisms by which odor information is encoded and processed in the olfactory system, the functional connectivity within and between olfactory brain areas, and the impact of spatiotemporal patterning of neuronal activity on higher-order neurons and behavioral outputs. The results give rise to a faceted picture of olfactory processing and provide insights into fundamental mechanisms underlying neuronal computations. This review focuses on some of this work presented in a Mini-Symposium at the Annual Meeting of the Society for Neuroscience in 2012.

A cupric silver histochemical analysis of domoic acid damage to olfactory pathways following status epilepticus in a rat model for chronic recurrent spontaneous seizures and aggressive behavior.

The amnesic shellfish toxin, domoic acid, interferes with glutamatergic pathways leading to neuronal damage, most notably causing memory loss and seizures. In this study, the authors utilized a recently developed rat model for domoic acid-induced epilepsy, an emerging disease appearing in California sea lions weeks to months after poisoning, to identify structural damage that may lead to a permanent epileptic state. Sprague Dawley rats were kindled with several low hourly intraperitoneal doses of domoic acid until a state of status epilepticus (SE) appears. This kindling approach has previously been shown to induce a permanent state of epileptic disease in 96% animals within 6 months. Three animals were selected for neurohistology a week after the initial SE. An amino cupric silver staining method using neutral red counterstain was used on every eighth 40 µm coronal section from each brain to highlight neural degeneration from the olfactory bulb through the brain stem. The most extensive damage was found in the olfactory bulb and related olfactory pathways, including the anterior/medial olfactory cortices, endopiriform nucleus, and entorhinal cortex. These findings indicate that damage to olfactory pathways is prominent in a rat model for domoic acid-induced chronic recurrent spontaneous seizures and aggressive behavior.

Different profiles of main and accessory olfactory bulb mitral/tufted cell projections revealed in mice using an anterograde tracer and a whole-mount, flattened cortex preparation.

A whole-mount, flattened cortex preparation was developed to compare profiles of axonal projections from main olfactory bulb (MOB) and accessory olfactory bulb (AOB) mitral and tufted (M/T) cells. After injections of the anterograde tracer, Phaseolus vulgaris leucoagglutinin, mapping of labeled axons using a Neurolucida system showed that M/T cells in the AOB sent axons primarily to the medial and posterior lateral cortical amygdala, with minimal branching into the piriform cortex. By contrast, M/T cells in the MOB displayed a network of collaterals that branched off the primary axon at several levels of the lateral olfactory tract (LOT). Collaterals emerging from the LOT into the anterior piriform cortex were often observed crossing into the posterior piriform cortex. M/T cells in the dorsal MOB extended fewer collaterals from the primary axon in the rostral LOT than did M/T cells from the anterior or ventral MOB. MOB M/T cells that projected to the medial amygdala did not do so exclusively, also sending collaterals to the anterior cortical amygdala as well as to olfactory cortical regions. This arrangement may be related to the ability of social experience to modify the response of mice to volatile pheromones detected by the main olfactory system.

Distribution of major brain gangliosides in olfactory tract of frogs.

Gangliosides are major cell-surface determinants in the central nervous system (CNS) of vertebrates, found both in neuronal and glial cell membranes. Together with cholesterol and glycosylphosphatidylinositol (GPI) - anchored proteins, gangliosides are involved in organization of plasma membrane microdomains. Based on biochemical studies, frog brain was previously described as having low quantities of gangliosides and their distribution pattern in specific brain regions was unknown. Using highly specific monoclonal antibodies generated against four major brain gangliosides (GM1, GD1a, GD1b and GT1b), we examined the distribution of these molecules in CNS of four different species of frogs (Rana esculenta, Rana temporaria, Bufo bufo and Bufo viridis). We also studied the distribution of myelin- associated glycoprotein (MAG), an inhibitor of axonal regeneration, which is a ligand for gangliosides GD1a and GT1b. Our results show that ganglioside GDla is expressed in neurons of olfactory bulb in all studied animals. In the brain of Rana sp., GD1a is expressed in the entire olfactory pathway, from olfactory bulbs to amygdala, while in Bufo sp. GD1a is restricted to the main olfactory bulb. Furthermore, we found that most of myelinated pathways in frogs express MAG, but do not express GD1a, which could be one of the reasons for better axon regeneration of neural pathways after CNS injury in amphibians in comparison to mammals.

Structural basis for noradrenergic regulation of neural circuits in the mouse olfactory bulb.

Olfactory input is processed in the glomerulus of the main olfactory bulb (OB) and relayed to higher centers in the brain by projection neurons. Conversely, centrifugal inputs from other brain regions project to the OB. We have previously analyzed centrifugal inputs into the OB from several brain regions using single-neuron labeling. In this study, we analyzed the centrifugal noradrenergic (NA) fibers derived from the locus coeruleus (LC), because their projection pathways and synaptic connections in the OB have not been clarified in detail. We analyzed the NA centrifugal projections by single-neuron labeling and immunoelectron microscopy. Individual NA neurons labeled by viral infection were three-dimensionally traced using Neurolucida software to visualize the projection pathway from the LC to the OB. Also, centrifugal NA fibers were visualized using an antibody for noradrenaline transporter (NET). NET immunoreactive (-ir) fibers contained many varicosities and synaptic vesicles. Furthermore, electron tomography demonstrated that NET-ir fibers formed asymmetrical synapses of varied morphology. Although these synapses were present at varicosities, the density of synapses was relatively low throughout the OB. The maximal density of synapses was found in the external plexiform layer; about 17% of all observed varicosities contained synapses. These results strongly suggest that NA-containing fibers in the OB release NA from both varicosities and synapses to influence the activities of OB neurons. The present study provides a morphological basis for olfactory modulation by centrifugal NA fibers derived from the LC.

Olfactory system involvement in natural scrapie disease.

The olfactory system (OS) is involved in many infectious and neurodegenerative diseases, both human and animal, and it has recently been investigated in regard to transmissible spongiform encephalopathies. Previous assessments of nasal mucosa infection by prions following intracerebral challenge suggested a potential centrifugal spread along the olfactory nerve fibers of the pathological prion protein (PrP(Sc)). Whether the nasal cavity may be a route for centripetal prion infection to the brain has also been experimentally studied. With the present study, we wanted to determine whether prion deposition in the OS occurs also under field conditions and what type of anatomical localization PrP(Sc) might display there. We report here on detection by different techniques of PrP(Sc) in the nasal mucosa and in the OS-related brain areas of sheep affected by natural scrapie. PrP(Sc) was detected in the perineurium of the olfactory nerve bundles in the medial nasal concha and in nasal-associated lymphoid tissue. Olfactory receptor neurons did not show PrP(Sc) immunostaining. PrP(Sc) deposition was found in the brain areas of olfactory fiber projection, chiefly in the olfactory bulb and the olfactory cortex. The prevalent PrP(Sc) deposition patterns were subependymal, perivascular, and submeningeal. This finding, together with the discovery of an intense PrP(Sc) immunostaining in the meningeal layer of the olfactory nerve perineurium, at the border with the subdural space extension surrounding the nerve rootlets, strongly suggests a probable role of cerebrospinal fluid in conveying prion infectivity to the nasal submucosa.

Histamine and histidine decarboxylase in the olfactory system and brain of the common cuttlefish Sepia officinalis (Linnaeus, 1758).

Cephalopods are radically different from any other invertebrate. Their molluscan heritage, innovative nervous system, and specialized behaviors create a unique blend of characteristics that are sometimes reminiscent of vertebrate features. For example, despite differences in the organization and development of their nervous systems, both vertebrates and cephalopods use many of the same neurotransmitters. One neurotransmitter, histamine (HA), has been well studied in both vertebrates and invertebrates, including molluscs. While HA was previously suggested to be present in the cephalopod central nervous system (CNS), Scaros, Croll, and Baratte only recently described the localization of HA in the olfactory system of the cuttlefish Sepia officinalis. Here, we describe the location of HA using an anti-HA antibody and a probe for histidine decarboxylase (HDC), a synthetic enzyme for HA. We extended previous descriptions of HA in the olfactory organ, nerve, and lobe, and describe HDC staining in the same regions. We found HDC-positive cell populations throughout the CNS, including the optic gland and the peduncle, optic, dorso-lateral, basal, subvertical, frontal, magnocellular, and buccal lobes. The distribution of HA in the olfactory system of S. officinalis is similar to the presence of HA in the chemosensory organs of gastropods but is different than the sensory systems in vertebrates or arthropods. However, HA's widespread abundance throughout the rest of the CNS of Sepia is a similarity shared with gastropods, vertebrates, and arthropods. Its widespread use with differing functions across Animalia provokes questions regarding the evolutionary history and adaptability of HA as a transmitter.

GABAergic Input From the Basal Forebrain Promotes the Survival of Adult-Born Neurons in the Mouse Olfactory Bulb.

A unique feature of the olfactory system is the continuous generation and integration of new neurons throughout adulthood. Adult-born neuron survival and integration is dependent on activity and sensory experience, which is largely mediated by early synaptic inputs that adult-born neurons receive upon entering the olfactory bulb (OB). As in early postnatal development, the first synaptic inputs onto adult-born neurons are GABAergic. However, the specific sources of early synaptic GABA and the influence of specific inputs on adult-born neuron development are poorly understood. Here, we use retrograde and anterograde viral tracing to reveal robust GABAergic projections from the basal forebrain horizontal limb of the diagonal band of Broca (HDB) to the granule cell layer (GCL) and glomerular layer (GL) of the mouse OB. Whole-cell electrophysiological recordings indicate that these projections target interneurons in the GCL and GL, including adult-born granule cells (abGCs). Recordings from birth-dated abGCs reveal a developmental time course in which HDB GABAergic input onto abGCs emerges as the neurons first enter the OB, and strengthens throughout the critical period of abGC development. Finally, we show that removing GABAergic signaling from HDB neurons results in decreased abGC survival. Together these data show that GABAergic projections from the HDB synapse onto immature abGCs in the OB to promote their survival through the critical period, thus representing a source of long-range input modulating plasticity in the adult OB.

Regional differences in the severity of Lewy body pathology across the olfactory cortex.

We studied alpha-synuclein pathology in the rhinencephalon of ten cases of Parkinson's disease (PD) and twelve neurologically normal controls, of which seven had incidental Lewy bodies in the substantia nigra at autopsy and five had no pathological evidence of neurological disease. In all PD and incidental Lewy bodies cases, alpha-synuclein pathology was found in all five subregions of the primary olfactory cortex that were sampled, and amongst them the pathology was significantly more severe in the temporal division of the piriform cortex than in the frontal division of the piriform cortex, olfactory tubercle or anterior portions of the entorhinal cortex. The orbitofrontal cortex, which is an area of projection from the primary olfactory cortex, was affected in some cases but overall the alpha-synuclein pathology was less severe in this area than in the primary olfactory cortex. Because different areas of the rhinencephalon are likely to play different roles in olfaction and our data indicate a differential involvement by alpha-synuclein deposition of structures implicated in smell, future prospective studies investigating the pathophysiological basis of hyposmia in PD should consider to examine the areas of primary olfactory cortex separately.

Imaging of phosphatidylcholines in the adult rat brain using MALDI-TOF MS.

Phosphatidylcholines (PCs) are the most abundant constituents of lipid in the brain. PCs function as major structural components of cell membranes and as important sources for signaling molecules. In the brain, three kinds of PCs, dipalmitoyl PC, palmitoyloleoyl PC, and stearoyloleoyl PC have been reported to be major species. They have different chemical and biological characteristics depending on the length of alkyl chains and the degree of saturation, suggesting that the abundance of PCs might be important to keep specialized membrane structures in the brain, such as myelin and synaptic membranes. However, detailed imaging of PCs in the total rat brain has not done yet. Thus, using imaging technology by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), we investigated the total distribution of PC32:0, PC34:1, and PC36:1 in the rat brain. PC32:0 and PC34:1 were more abundantly observed in the gray matter areas than in the white matter areas throughout the central nervous system (CNS), while PC36:1 was evenly seen at low levels in both areas. In addition, we found that PC32:0 and PC34:1 were detected at very high levels in the granular layer of the olfactory bulb, piriform cortex, insular cortex, and molecular layer of the cerebellum, which are known for areas showing high neuronal plasticity. The present imaging data clearly show that various PCs are differentially distributed throughout the rat CNS, and suggest that these differential distributions of various PCs are necessary to keep normal brain functions.

Ric-8B interacts with G alpha olf and G gamma 13 and co-localizes with G alpha olf, G beta 1 and G gamma 13 in the cilia of olfactory sensory neurons.

Olfactory sensory neurons are able to detect odorants with high sensitivity and specificity. We have demonstrated that Ric-8B, a guanine nucleotide exchange factor (GEF), interacts with Galphaolf and enhances odorant receptor signaling. Here we show that Ric-8B also interacts with Ggamma13, a divergent member of the Ggamma subunit family which has been implicated in taste signal transduction, and is abundantly expressed in the cilia of olfactory sensory neurons. We show that Gbeta1 is the predominant Gbeta subunit expressed in the olfactory sensory neurons. Ric-8B and Gbeta1, like Galphaolf and Ggamma13, are enriched in the cilia of olfactory sensory neurons. We also show that Ric-8B interacts with Galphaolf in a nucleotide dependent manner, consistent with the role as a GEF. Our results constitute the first example of a GEF protein that interacts with two different olfactory G protein subunits and further implicate Ric-8B as a regulator of odorant signal transduction.

Neuropilin-2, a novel member of the neuropilin family, is a high affinity receptor for the semaphorins Sema E and Sema IV but not Sema III.

Semaphorins are a large family of secreted and transmembrane proteins, several of which are implicated in repulsive axon guidance. Neuropilin (neuropilin-1) was recently identified as a receptor for Collapsin-1/Semaphorin III/D (Sema III). We report the identification of a related protein, neuropilin-2, whose mRNA is expressed by developing neurons in a pattern largely, though not completely, nonoverlapping with that of neuropilin-1. Unlike neuropilin-1, which binds with high affinity to the three structurally related semaphorins Sema III, Sema E, and Sema IV, neuropilin-2 shows high affinity binding only to Sema E and Sema IV, not Sema III. These results identify neuropilins as a family of receptors (or components of receptors) for at least one semaphorin subfamily. They also suggest that the specificity of action of different members of this subfamily may be determined by the complement of neuropilins expressed by responsive cells.

Brain morphology and immunohistochemical localization of the gonadotropin-releasing hormone in the bluefin tuna, Thunnus thynnus.

The present study was focused on the morphology of the diencephalic nuclei (likely involved in reproductive functions) as well as on the distribution of GnRH (gonadotropin-releasing hormone) in the rhinencephalon, telencephalon and the diencephalon of the brain of bluefin tuna (Thunnus thynnus) by means of immunohistochemistry. Bluefin tuna has an encephalization quotient (QE) similar to that of other large pelagic fish. Its brain exhibits well-developed optic tecta and corpus cerebelli. The diencephalic neuron cell bodies involved in reproductive functions are grouped in two main nuclei: the nucleus preopticus-periventricularis and the nucleus lateralis tuberis. The nucleus preopticus-periventricularis consists of the nucleus periventricularis and the nucleus preopticus consisting of a few sparse multipolar neurons in the rostral part and numerous cells closely packed and arranged in several layers in its aboral part. The nucleus lateralis tuberis is located in the ventral-lateral area of the diencephalon and is made up of a number of large multipolar neurones. Four different polyclonal primary antibodies against salmon (s)GnRH, chicken (c)GnRH-II (cGnRH-II 675, cGnRH-II 6) and sea bream (sb)GnRH were employed in the immunohistochemical experiments. No immunoreactive structures were found with anti sbGnRH serum. sGnRH and cGnRH-II antisera revealed immunoreactivity in the perikarya of the olfactory bulbs, preopticus-periventricular nucleus, oculomotor nucleus and midbrain tegmentum. The nucleus lateralis tuberis showed immunostaining only with anti-sGnRH serum. Nerve fibres immunoreactive to cGnRH and sGnRH sera were found in the olfactory bulbs, olfactory nerve and neurohypophysis. The significance of the distribution of the GnRH-immunoreactive neuronal structures is discussed.

A Mirror-Symmetric Excitatory Link Coordinates Odor Maps across Olfactory Bulbs and Enables Odor Perceptual Unity.

Sensory input reaching the brain from bilateral and offset channels is nonetheless perceived as unified. This unity could be explained by simultaneous projections to both hemispheres, or inter-hemispheric information transfer between sensory cortical maps. Odor input, however, is not topographically organized, nor does it project bilaterally, making olfactory perceptual unity enigmatic. Here we report a circuit that interconnects mirror-symmetric isofunctional mitral/tufted cells between the mouse olfactory bulbs. Connected neurons respond to similar odors from ipsi- and contra-nostrils, whereas unconnected neurons do not respond to odors from the contralateral nostril. This connectivity is likely mediated through a one-to-one mapping from mitral/tufted neurons to the ipsilateral anterior olfactory nucleus pars externa, which activates the mirror-symmetric isofunctional mitral/tufted neurons glutamatergically. This circuit enables sharing of odor information across hemispheres in the absence of a cortical topographical organization, suggesting that olfactory glomerular maps are the equivalent of cortical sensory maps found in other senses.

Activity-dependent plasticity in the olfactory intrabulbar map.

In mammals, each olfactory bulb contains two mirror-symmetric glomerular maps. Isofunctional glomeruli within each bulb are specifically linked through a set of reciprocal intrabulbar projections (IBPs) to form an intrabulbar map. We injected neural tracers into the glomerular layer on one side of the bulb and examined the resulting projection on the opposite side. In adult mice, the size of the projection tuft is directly proportional to the size of the injected region. Using this ratio as a measure of IBP maturity, we find an immature 5:1 projection to injection ratio at 1 week of age that gradually refines to a mature 1:1 by 7 weeks. Moreover, whereas the glomerular map is able to form despite the elimination of odorant-induced activity, the intrabulbar map shows clear activity dependence for its precise formation. Here we show through experiments with both naris-occluded and anosmic mice that odorant-induced activity is not required to establish IBPs but is crucial for projection refinement. In contrast, increased glomerular activation through exposure to distinct odorants during map development can accelerate the refinement of projections associated with the activated glomeruli. These findings illustrate a clear role for odorant-induced activity in shaping the internal circuitry of the bulb. Interestingly, activity deprivation can alter the organization of both the developing and the mature map to the same degree, demonstrating that intrabulbar map plasticity is maintained into adulthood with no discernible critical period.

Relation of chemical structure to specificity of response in olfactory glomeruli.

Recent work on the mammalian olfactory system shows that sensory neurons expressing the same type of odor receptor converge their axons onto one or a few glomeruli. This nearly one-to-one correspondence between an odor receptor type and an olfactory glomerulus gives rise to the tuning specificity of bulbar mitral and tufted cells, such that individual cells are only activated by a range of odor molecules having a similar chemical structure. These findings now make it possible to analyze the molecular mechanisms involved in the functional differentiation of sensory neurons, as well as those involved in their specific connections with bulbar neurons.