The neurovascular unit of capillary blood vessels in the rat nervous system. A rapid-Golgi electron microscopy study.

We describe a pericapillary organ in the rat forebrain and cerebellar cortex. It consists of a series of tripartite synapses with synaptic extensions enveloped by astrocytic endfeet that are linked to the capillary wall by synaptic extensions. Reciprocal specializations of the pericyte-capillary blood vessel (CBV) with such specialized synapses suggest a mechanoreceptor role. In Golgi-impregnated and 3D reconstructions of the cerebral cortex and thalamus, a series of TSs appear to be sequentially ordered in a common dendrite, paralleled by synaptic outgrowths termed golf club synaptic extensions (GCE) opposed to a longitudinal crest (LC) from the capillary basal lamina (BL). Our results show that, in the cerebellar cortex, afferent fibers and interneurons display microanatomical structures that strongly suggest an interaction with the capillary wall. Afferent mossy fiber (MF) rosettes and ascending granule cell axons and their dendrites define the pericapillary passage interactions that are entangled by endfeet. The presence of mRNA of the mechanosensitive channel Piezo1 in the MF rosettes, together with the surrounding end-feet and the capillary wall form mechanosensory units. The ubiquity of such units to modulate synaptic transmission is also supported by Piezo1 mRNA expressing pyramidal isocortical and thalamic neurons. This scenario suggests that ascending impulses to the cerebellar and cortical targets are presynaptically modulated by the reciprocal interaction with the mechanosensory pericapillary organ that ultimately modulates the vasomotor response.

Multi-tensor diffusion abnormalities of gray matter in an animal model of cortical dysplasia.

Focal cortical dysplasias are a type of malformations of cortical development that are a common cause of drug-resistant focal epilepsy. Surgical treatment is a viable option for some of these patients, with their outcome being highly related to complete surgical resection of lesions visible in magnetic resonance imaging (MRI). However, subtle lesions often go undetected on conventional imaging. Several methods to analyze MRI have been proposed, with the common goal of rendering subtle cortical lesions visible. However, most image-processing methods are targeted to detect the macroscopic characteristics of cortical dysplasias, which do not always correspond to the microstructural disarrangement of these cortical malformations. Quantitative analysis of diffusion-weighted MRI (dMRI) enables the inference of tissue characteristics, and novel methods provide valuable microstructural features of complex tissue, including gray matter. We investigated the ability of advanced dMRI descriptors to detect diffusion abnormalities in an animal model of cortical dysplasia. For this purpose, we induced cortical dysplasia in 18 animals that were scanned at 30 postnatal days (along with 19 control animals). We obtained multi-shell dMRI, to which we fitted single and multi-tensor representations. Quantitative dMRI parameters derived from these methods were queried using a curvilinear coordinate system to sample the cortical mantle, providing inter-subject anatomical correspondence. We found region- and layer-specific diffusion abnormalities in experimental animals. Moreover, we were able to distinguish diffusion abnormalities related to altered intra-cortical tangential fibers from those associated with radial cortical fibers. Histological examinations revealed myelo-architectural abnormalities that explain the alterations observed through dMRI. The methods for dMRI acquisition and analysis used here are available in clinical settings and our work shows their clinical relevance to detect subtle cortical dysplasias through analysis of their microstructural properties.

The olfactory limbus of the red fox (Vulpes vulpes). New insights regarding a noncanonical olfactory bulb pathway.

Introduction: The olfactory system in most mammals is divided into several subsystems based on the anatomical locations of the neuroreceptor cells involved and the receptor families that are expressed. In addition to the main olfactory system and the vomeronasal system, a range of olfactory subsystems converge onto the transition zone located between the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), which has been termed the olfactory limbus (OL). The OL contains specialized glomeruli that receive noncanonical sensory afferences and which interact with the MOB and AOB. Little is known regarding the olfactory subsystems of mammals other than laboratory rodents. Methods: We have focused on characterizing the OL in the red fox by performing general and specific histological stainings on serial sections, using both single and double immunohistochemical and lectin-histochemical labeling techniques. Results: As a result, we have been able to determine that the OL of the red fox (Vulpes vulpes) displays an uncommonly high degree of development and complexity. Discussion: This makes this species a novel mammalian model, the study of which could improve our understanding of the noncanonical pathways involved in the processing of chemosensory cues.

Contributions of Lorente de Nó to the Nonlinear Circuitry of the Brain.

Rafael Lorente de Nó, the youngest disciple of Santiago Ramón y Cajal, made significant and versatile contributions to the broad area of neuroscience. Present assay highlights the groundbreaking contributions of this Spanish investigator to neuronal connectivity. From Lorente de Nó laws of plurality and recurrence of connections among neurons emerged nonlinear connectivity and, therefore, set the foundation to understand the emergent properties of neuronal circuits. The emergence, truthfulness, and applicability of these organizing principles are discussed in the context of their current impact in studying neuronal ensembles. Anat Rec, 303:1215-1220, 2020. © 2019 American Association for Anatomy.

Histological and diffusion-weighted magnetic resonance imaging data from normal and degenerated optic nerve and chiasm of the rat.

Diffusion-weighted magnetic resonance imaging (dMRI) is widely used to infer microstructural characteristics of tissue, particularly in cerebral white matter. Histological validation of the metrics derived from dMRI methods are needed to fully characterize their ability to capture biologically-relevant histological features non-invasively. The data described here were used to correlate metrics derived from dMRI and quantitative histology in an animal model of axonal degeneration ("Histological validation of per-bundle water diffusion metrics within a region of fiber crossing following axonal degeneration" [1]). Unilateral retinal ischemia/reperfusion was induced in 10 rats, by the elevation of pressure of the anterior chamber of the eye for 90 min. Five rats were used as controls. After five weeks, injured animals were intracardially perfused to analyze the optic nerves and chiasm with dMRI and histology. This resulted in 15 brain scans, each with 80 diffusion-sensitizing gradient directions with b = 2000 and 2500 s/mm2 and 20 non-diffusion-weighted images (b = 0 s/mm2), with isometric voxel resolution of 125 µm3. Histological sections were obtained after dMRI. Optical microscopy photomicrographs of the optic nerves (stained with toluidine blue) are available, as well as their corresponding automatic segmentations of axons and myelin.

Histological validation of per-bundle water diffusion metrics within a region of fiber crossing following axonal degeneration.

Micro-architectural characteristics of white matter can be inferred through analysis of diffusion-weighted magnetic resonance imaging (dMRI). The diffusion-dependent signal can be analyzed through several methods, with the tensor model being the most frequently used due to its straightforward interpretation and low requirements for acquisition parameters. While valuable information can be gained from the tensor-derived metrics in regions of homogeneous tissue organization, this model does not provide reliable microstructural information at crossing fiber regions, which are pervasive throughout human white matter. Several multiple fiber models have been proposed that seem to overcome the limitations of the tensor, with few providing per-bundle dMRI-derived metrics. However, biological interpretations of such metrics are limited by the lack of histological confirmation. To this end, we developed a straightforward biological validation framework. Unilateral retinal ischemia was induced in ten rats, which resulted in axonal (Wallerian) degeneration of the corresponding optic nerve, while the contralateral was left intact; the intact and injured axonal populations meet at the optic chiasm as they cross the midline, generating a fiber crossing region in which each population has different diffusion properties. Five rats served as controls. High-resolution ex vivo dMRI was acquired five weeks after experimental procedures. We correlated and compared histology to per-bundle descriptors derived from three methodologies for dMRI analysis (constrained spherical deconvolution and two multi-tensor representations). We found a tight correlation between axonal density (as evaluated through automatic segmentation of histological sections) with per-bundle apparent fiber density and fractional anisotropy (derived from dMRI). The multi-fiber methods explored were able to correctly identify the damaged fiber populations in a region of fiber crossings (chiasm). Our results provide validation of metrics that bring substantial and clinically useful information about white-matter tissue at crossing fiber regions. Our proposed framework is useful to validate other current and future dMRI methods.

On the existence of mechanoreceptors within the neurovascular unit of the mammalian brain.

We describe a set of perivascular interneurons (PINs) with series of fibro-vesicular complexes (FVCs) throughout the gray matter of the adult rabbit and rat brains. PIN-FVCs are ubiquitous throughout the brain vasculature as detected in Golgi-impregnated specimens. Most PINs are small, aspiny cells with short or long (> 1 mm) axons that split and travel along arterial blood vessels. Upon ramification, axons form FVCs around the arising vascular branches; then, paired axons run parallel to the vessel wall until another ramification ensues, and a new FVC is formed. Cytologically, FVCs consist of clusters of perivascular bulbs (PVBs) encircling the precapillary and capillary wall surrounded by end-feet and the extracellular matrix of endothelial cells and pericytes. A PVB contains mitochondria, multivesicular bodies, and granules with a membranous core, similar to Meissner corpuscles and other mechanoreceptors. Some PVBs form asymmetrical, axo-spinous synapses with presumptive adjacent neurons. PINs appear to correspond to the type 1 nNOS-positive neurons whose FVCs co-label with markers of sensory fiber-terminals surrounded by astrocytic end-feet. The PIN is conserved in adult cats and rhesus monkey specimens. The location, ubiquity throughout the vasculature of the mammalian brain, and cytological organization of the PIN-FVCs suggests that it is a sensory receptor intrinsic to the mammalian neurovascular unit that corresponds to an afferent limb of the sensorimotor feed-back mechanism controlling local blood flow.

Olfaction and Pheromones: Uncanonical Sensory Influences and Bulbar Interactions.

The rodent main and accessory olfactory systems (AOS) are considered functionally and anatomically segregated information-processing pathways. Each system is devoted to the detection of volatile odorants and pheromones, respectively. However, a growing number of evidences supports a cooperative interaction between them. For instance, at least four non-canonical receptor families (i.e., different from olfactory and vomeronasal receptor families) have been recently discovered. These atypical receptor families are expressed in the sensory organs of the nasal cavity and furnish parallel processing-pathways that detect specific stimuli and mediate specific behaviors as well. Aside from the receptor and functional diversity of these sensory modalities, they converge into a poorly understood bulbar area at the intersection of the main- main olfactory bulb (MOB) and accessory olfactory bulb (AOB) that has been termed olfactory limbus (OL). Given the intimate association the OL with specialized glomeruli (i.e., necklace and modified glomeruli) receiving uncanonical sensory afferences and its interactions with the MOB and AOB, the possibility that OL is a site of non-olfactory and atypical vomeronasal sensory decoding is discussed.

Is There Evidence for Myelin Modeling by Astrocytes in the Normal Adult Brain?

A set of astrocytic process associated with altered myelinated axons is described in the forebrain of normal adult rodents with confocal, electron microscopy, and 3D reconstructions. Each process consists of a protuberance that contains secretory organelles including numerous lysosomes which polarize and open next to disrupted myelinated axons. Because of the distinctive asymmetric organelle distribution and ubiquity throughout the forebrain neuropil, this enlargement is named paraxial process (PAP). The myelin envelope contiguous to the PAP displays focal disruption or disintegration. In routine electron microscopy clusters of large, confluent, lysosomes proved to be an effective landmark for PAP identification. In 3D assemblies lysosomes organize a series of interconnected saccules that open up to the plasmalemma next to the disrupted myelin envelope(s). Activity for acid hydrolases was visualized in lysosomes, and extracellularly at the PAP-myelin interface and/or between the glial and neuronal outer aspects. Organelles in astrocytic processes involved in digesting pyknotic cells and debris resemble those encountered in PAPs supporting a likewise lytic function of the later. Conversely, processes entangling tripartite synapses and glomeruli were devoid of lysosomes. Both oligodendrocytic and microglial processes were not associated with altered myelin envelopes. The possible roles of the PAP in myelin remodeling in the context of the oligodendrocyte-astrocyte interactions and in the astrocyte's secretory pathways are discussed.

Electrophysiological Evidence for a Direct Link between the Main and Accessory Olfactory Bulbs in the Adult Rat.

It is accepted that the main- and accessory- olfactory systems exhibit overlapping responses to pheromones and odorants. We performed whole-cell patch-clamp recordings in adult rat olfactory bulb slices to define a possible interaction between the first central relay of these systems: the accessory olfactory bulb (AOB) and the main olfactory bulb (MOB). This was tested by applying electrical field stimulation in the dorsal part of the MOB while recording large principal cells (LPCs) of the anterior AOB (aAOB). Additional recordings of LPCs were performed at either side of the plane of intersection between the aAOB and posterior-AOB (pAOB) halves, or linea alba, while applying field stimulation to the opposite half. A total of 92 recorded neurons were filled during whole-cell recordings with biocytin and studied at the light microscope. Neurons located in the aAOB (n = 6, 8%) send axon collaterals to the MOB since they were antidromically activated in the presence of glutamate receptor antagonists (APV and CNQX). Recorded LPCs evoked orthodromic excitatory post-synaptic responses (n = 6, aAOB; n = 1, pAOB) or antidromic action potentials (n = 8, aAOB; n = 7, pAOB) when applying field stimulation to the opposite half of the recording site (e.g., recording in aAOB; stimulating in pAOB, and vice-versa). Observation of the filled neurons revealed that indeed, LPCs send axon branches that cross the linea alba to resolve in the internal cellular layer. Additionally, LPCs of the aAOB send axon collaterals to dorsal-MOB territory. Notably, while performing AOB recordings we found a sub-population of neurons (24% of the total) that exhibited voltage-dependent bursts of action potentials. Our findings support the existence of: 1. a direct projection from aAOB LPCs to dorsal-MOB, 2. physiologically active synapses linking aAOB and pAOB, and 3. pacemaker-like neurons in both AOB halves. This work was presented in the form of an Abstract on SfN 2014 (719.14/EE17).

Some predictions of Rafael Lorente de Nó 80 years later.

Rafael Lorente de Nó, the youngest of Santiago Ramón y Cajal disciples, was one of the last Century's more influential researches in neuroscience. This assay highlights two fundamental contributions of Rafael Lorente de Nó to neurobiology: the intrinsic organization of the mammalian cerebral cortex and the basic physiology of the neuron processes.

Structural variation and interactions among astrocytes of the rostral migratory stream and olfactory bulb: II. Golgi and electron microscopic study of the adult rat.

Cytological characteristics of a cell sharing structure of both an astrocyte and a neuron, previously termed amphomorphic cell (AC), were defined here in adult rat rostral migratory stream (RMS). The AC perikaryon corresponds to that of the B1 cell of the adult mouse subventricular zone (SVZ)-RMS. The AC and its processes are confined to the RMS. Each AC originates four sets of processes that overlap with those from its homologues and adjacent neural and stromal elements. ACs interact between them via reciprocal sets of processes: those directed caudally bear spheroidal vesicles (SVP) and form gap junctions with pleomorphic vesicles (PVs) associated with the anterior set from the adjacent AC. Large asymmetric synapses, a set of them arising from the anterior olfactory nucleus, converge on each SV. The interlacing processes of the AC, together with a set of perikaryal out-growths form the glial cuff surrounding migrating neuroblasts described earlier. Small asymmetrical and symmetrical synapses terminate in subsets of differentiated A-cells, termed here A1, in the bulbar part of the RMS. Both AC- and A1-cells form electrical synapses between them and with their homologues. The strategic, wide-spread distribution between the neuropil and blood vessels of the AC, its processes, and migrating neuroblasts, suggests that the AC might mediate between both endogenous inductors and neurotransmitters, influencing the adult-born neurons it had previously originated.

Structural variation and interactions among astrocytes of the rostral migratory stream and olfactory bulb of the adult rabbit. I. Light microscopic study.

The organization of the adult rabbit rostral migratory stream (RMS) was studied utilizing the rapid-Golgi technique and aniline stains. The RMS consists of a bi-layered, concentric structure bounding the olfactory ventricle. The inner hypercellular layer contains numerous small- to medium-sized cells and is encased by the stratum lucidum (SL), a layer of moderate to low cell density. The most frequent cell in the SL, here termed an amphomorphic cell (AC), has a mixed astrocyte-neuron phenotype. Although each AC exhibits four sets of processes or domains, variation in the complexity of these domains justified dividing them into six subclasses that might correspond to successive stages of differentiation described earlier in rodents.

A cytological and experimental study of the neuropil and primary olfactory afferences to the piriform cortex.

The microscopic organization of the piriform cortex (PC) was studied in normal and experimental material from adult albino rats. In rapid-Golgi specimens a set of collaterals from the lateral olfactory tract (i.e., sublayer Ia) to the neuropil of the Layer II (LII) was identified. Specimens from experimental animals that received electrolytic lesion of the main olfactory bulb three days before sacrificing, were further processed for pre-embedding immunocytochemistry to the enzyme glutamic acid decarboxylase 67 (GAD 67). This novel approach permitted a simultaneous visualization at electron microscopy of both synaptic degeneration and GAD67-immunoreactive (GAD-I) sites. Degenerating and GAD-I synapses were separately found in the neuropil of Layers I and II of the PC. Previously overlooked patches of neuropil were featured in sublayer Ia. These areas consisted of dendritic and axonal processes including four synaptic types. Tridimensional reconstructions from serial thin sections from LI revealed the external appearance of the varicose and tubular dendrites as well as the synaptic terminals therein. The putative source(s) of processes to the neuropil of sublayer Ia is discussed in the context of the internal circuitry of the PC and an alternative model is introduced.

Functional impact of interneuronal inhibition in the cerebral cortex of behaving animals.

This paper reviews recent progress in understanding the functional roles of inhibitory interneurons in behaving animals and how they affect information processing in cortical microcircuits. Multiple studies have shown that the morphological subtypes of inhibitory cells show distinct electrophysiological properties, as well as different molecular and neurochemical identities, providing a large mosaic of inhibitory mechanisms for the dynamic processing of information in the cortex. However, it is only recently that some specific functions of different interneuronal subtypes have been described in behaving animals. In this regard, influential results have been obtained using the known differences of interneurons and pyramidal cells recorded extracellularly to dissociate the functional roles that these two classes of neurons may play in the cortical microcircuits during various behaviors. Neurons can be segregated into fast-spiking (FS) cells that show short action potentials, high discharge rates, and correspond to putative interneurons; and regular-spiking (RS) cells that show larger action potentials and correspond to pyramidal neurons. Using this classification strategy, it has been found that cortical inhibition is involved in sculpting the tuning to different stimulus or behavioral features across a wide variety of sensory, association, and motor areas. Recent studies have suggested that the increase in high-frequency synchronization during information processing and spatial attention may be mediated by FS activation. Finally, FS are active during motor planning and movement execution in different motor areas, supporting the notion that inhibitory interneurons are involved in shaping the motor command but not in gating the cortical output.

Cytological organization of the alpha component of the anterior olfactory nucleus and olfactory limbus.

This study describes the microscopic organization of a wedge-shaped area at the intersection of the main (MOB) and accessory olfactory bulbs (AOBs), or olfactory limbus (OL), and an additional component of the anterior olfactory nucleus or alpha AON that lies underneath of the AOB. The OL consists of a modified bulbar cortex bounded anteriorly by the MOB and posteriorly by the AOB. In Nissl-stained specimens the OL differs from the MOB by a progressive, antero-posterior decrease in thickness or absence of the external plexiform, mitral/tufted cell, and granule cell layers. On cytoarchitectual grounds the OL is divided from rostral to caudal into three distinct components: a stripe of glomerular-free cortex or preolfactory area (PA), a second or necklace glomerular area, and a wedge-shaped or interstitial area (INA) crowned by the so-called modified glomeruli that appear to belong to the anterior AOB. The strategic location and interactions with the main and AOBs, together with the previously noted functional and connectional evidence, suggest that the OL may be related to both sensory modalities. The alpha component of the anterior olfactory nucleus, a slender cellular cluster (i.e., 650 × 150 µm) paralleling the base of the AOB, contains two neuron types: a pyramidal-like neuron and an interneuron. Dendrites of pyramidal-like cells (P-L) organize into a single bundle that ascends avoiding the AOB to resolve in a trigone bounded by the edge of the OL, the AOB and the dorsal part of the anterior olfactory nucleus. Utrastructurally, the neuropil of the alpha component contains three types of synaptic terminals; one of them immunoreactive to the enzyme glutamate decarboxylase, isoform 67.

Paced-mating increases the number of adult new born cells in the internal cellular (granular) layer of the accessory olfactory bulb.

The continuous production and addition of new neurons during life in the olfactory bulb is well accepted and has been extensively studied in rodents. This process could allow the animals to adapt to a changing environment. Olfactory neurogenesis begins in the subventricular zone where stem cells proliferate and give rise to young undifferentiated neuroblasts that migrate along the rostral migratory stream to the olfactory bulb (OB). Olfaction is crucial for the expression of sexual behavior in rodents. In female rats, the ability to control the rate of sexual interactions (pacing) has important physiological and behavioral consequences. In the present experiment we evaluated if pacing behavior modifies the rate of new cells that reach the main and accessory olfactory bulb. The BrdU marker was injected before and after different behavioral tests which included: females placed in a mating cage (control), females allowed to pace the sexual interaction, females that mated but were not able to control the rate of the sexual interaction and females exposed to a sexually active male. Subjects were sacrificed fifteen days after the behavioral test. We observed a significant increase in the density of BrdU positive cells in the internal cellular layer of the accessory olfactory bulb when females paced the sexual interaction in comparison to the other 3 groups. No differences in the cell density in the main olfactory bulb were found. These results suggest that pacing behavior promotes an increase in density of the new cells in the accessory olfactory bulb.