Cell Body Layer Research Articles

Neuronal segmentation in cephalopod arms

Prehensile arms are among the most remarkable features of the octopus, but little is known about the neural circuitry controlling arm movements. Here, we report on the cellular and molecular organization of the arm nervous system, focusing on its massive axial nerve cords (ANCs). We found that the ANC is segmented. In transverse cross sections, the ANC cell body layer wraps around the neuropil with no apparent segregation of sensory and motor neurons. In longitudinal sections, however, ANC neurons form segments, setting up a modular organization to the adjoining neuropil. ANC nerves exit in the septa between segments, and for each sucker, form a spatial topographic map (“suckerotopy”). A strong link between ANC segmentation and flexible sucker-laden arms is confirmed by comparative study of squid arms and tentacles. These ANC modules offer a template for modeling the motor control of soft tissues and provide a compelling example of nervous system segmentation in molluscs.

Repeating ultrastructural motifs provide insight into the organization of the octopus arm nervous system

The peripheral nervous system of the octopus is among the most complex of any animal. In each arm, hundreds of serial ganglia form a central core of nervous tissue processing sensory input, issuing motor commands, and exchanging information with the central brain.1,2,3,4,5 In addition to the central cord, there are two other types of neural elements: fine intramuscular nerve cords (INCs)6,7 and small sucker ganglia at the base of each sucker.2,6,8,9 Connections between these different elements and the structural organization of the arm nervous system remain poorly understood, despite decades of interest and a more recent explosion of studies of the cephalopod nervous system.8,10,11,12,13,14,15 Here, we use serial blockface electron microscopy to reconstruct large volumes of an arm from Octopus bocki at the base and toward the tip, mapping connections between the various neural elements and their relationship to the muscle and skin. We show that the ganglia follow an alternating mirror-image pattern along the arm, where the left or right-sided location of successive suckers determines ganglionic orientation. We also describe previously unrecognized patterns in (1) continuity of oblique connectives between the INCs that encircle the arm; (2) repeatable structures of the major blood vessel branches and nerve connectives within each ganglion; (3) clustering of rare, unusually large neurons within the cell body layers; and (4) division of the cortex into repeating columns. These new findings from the first 3DEM reconstruction of the arm should greatly facilitate future studies of octopus neurobiology, particularly sensori-motor integration and arm control.

Neuronal segmentation in cephalopod arms.

The prehensile arms of the cephalopod are among these animals' most remarkable features, but little is known about the neural circuitry governing arm and sucker movements1,2. Here, we investigated the cellular and molecular organization of the arm nervous system, focusing on the massive axial nerve cords (ANCs) in the octopus arms which collectively harbor four times as many neurons as the central brain3. We found that the ANC is segmented. In transverse cross sections, the ANC cell body layer wraps around the neuropil with no apparent segregation of sensory and motor neurons. In longitudinal sections, however, ANC neurons form segments, setting up a modular organization to the adjoining ANC neuropil. The septa between each segment are, in contrast, neuron-poor but contain nerve exits, vasculature and abundant collagen. Surprisingly, nerves exiting from neighboring septa differ in their fiber trajectories indicating that multiple adjoining segments must cooperate to innervate the arm musculature fully. The nerves for each sucker also exit through septa and set up a spatial "suckerotopy" in the ANC. A strong link between ANC segmentation and flexible sucker-laden arms was confirmed by comparative study of squid arms and tentacles. The ANC segmental modules represent a new template for understanding the motor control of octopus soft tissues. They also provide the first example of nervous system segmentation in a mollusc4.

Neuronal segmentation in cephalopod arms.

The prehensile arms of the cephalopod are among these animals most remarkable features, but the neural circuitry governing arm and sucker movements remains largely unknown. We studied the neuronal organization of the adult axial nerve cord (ANC) of Octopus bimaculoides with molecular and cellular methods. The ANCs, which lie in the center of every arm, are the largest neuronal structures in the octopus, containing four times as many neurons as found in the central brain. In transverse cross section, the cell body layer (CBL) of the ANC wraps around its neuropil (NP) with little apparent segregation of sensory and motor neurons or nerve exits. Strikingly, when studied in longitudinal sections, the ANC is segmented. ANC neuronal cell bodies form columns separated by septa, with 15 segments overlying each pair of suckers. The segments underlie a modular organization to the ANC neuropil: neuronal cell bodies within each segment send the bulk of their processes directly into the adjoining neuropil, with some reaching the contralateral side. In addition, some nerve processes branch upon entering the NP, forming short-range projections to neighboring segments and mid-range projections to the ANC segments of adjoining suckers. The septa between the segments are employed as ANC nerve exits and as channels for ANC vasculature. Cellular analysis establishes that adjoining septa issue nerves with distinct fiber trajectories, which across two segments (or three septa) fully innervate the arm musculature. Sucker nerves also use the septa, setting up a nerve fiber "suckerotopy" in the sucker-side of the ANC. Comparative anatomy suggests a strong link between segmentation and flexible sucker-laden arms. In the squid Doryteuthis pealeii, the arms and the sucker-rich club of the tentacles have segments, but the sucker-poor stalk of the tentacles does not. The neural modules described here provide a new template for understanding the motor control of octopus soft tissues. In addition, this finding represents the first demonstration of nervous system segmentation in a mollusc.

Retinal clues for selective neuronal loss in multiple sclerosis.

The relationship between the cell body layer and the dendritic network layer of the retina and cognitive performance (CP) in MS patients has not been examined separately. The objective of this study is to predict cognitive impairment (CI) in RRMS patients and to examine the relationship between CP and ganglion cell layer (GCL), inner plexiform layer (IPL), and GCL divided by IPL (GCL/IPL). Ophthalmological evaluation, retinal segmentation, and Symbol Digit Modalities Test (SDMT) were performed on 102 RRMS patients and 54 healthy subjects. The relationships of GCL, IPL, and GCL/IPL with CP in eyes without a history of optic neuritis were investigated using Spearman's correlation. Models were created by accepting 1 standard deviation less of the SDMT mean of the control group as the limit for CI. The cutoff value of the GCL/IPL variable that could predict CI was calculated by ROC analysis, and the ability to accurately predict CI was tested with binary logistic regression. No correlation was found between OCT parameters and CP in healthy subjects. Correlation was found between GCL thickness and GCL/IPL variable and CP in RRMS patients (r=0.235, r=0.667 respectively). A GCL/IPL value of 1.255 was able to identify CI with 81.8% sensitivity and 75.9% specificity (AUC=0.844, LR=3.38) and predicted CI with 74.5% accuracy (Nagelkerke R2=0.439). In RRMS patients, the IPL thickness is unrelated to CP. Therewithal, the GCL/IPL-CP relationship is stronger than the GCL-CP relationship and GCL/IPL can predict CI.

Enhanced presynaptic mitochondrial energy production is required for memory formation

Some of the prominent features of long-term memory formation include protein synthesis, gene expression, enhanced neurotransmitter release, increased excitability, and formation of new synapses. As these processes are critically dependent on mitochondrial function, we hypothesized that increased mitochondrial respiration and dynamics would play a prominent role in memory formation. To address this possibility, we measured mitochondrial oxygen consumption (OCR) in hippocampal tissue punches from trained and untrained animals. Our results show that context fear training significantly increased basal, ATP synthesis-linked, and maximal OCR in the Shaffer collateral-CA1 synaptic region, but not in the CA1 cell body layer. These changes were recapitulated in synaptosomes isolated from the hippocampi of fear-trained animals. As dynamin-related protein 1 (Drp1) plays an important role in mitochondrial fission, we examined its role in the increased mitochondrial respiration observed after fear training. Drp1 inhibitors decreased the training-associated enhancement of OCR and impaired contextual fear memory, but did not alter the number of synaptosomes containing mitochondria. Taken together, our results show context fear training increases presynaptic mitochondria respiration, and that Drp-1 mediated enhanced energy production in CA1 pre-synaptic terminals is necessary for context fear memory that does not result from an increase in the number of synaptosomes containing mitochondria or an increase in mitochondrial mass within the synaptic layer.

Architecture of the inferior parietal cortex in capuchin monkey.

We studied the organization of the inferior parietal cortex (IPC) in five capuchin monkey (6 hemispheres) using cytoarchitectonic (Nissl), myeloarchitectonic (Gallyas), and immune-architectonic (SMI-32 monoclonal antibody) techniques. We partitioned the IPC into five distinct areas: PFG, PG, Opt, PFop, and PGop. Since we used parasagittal sections, we were not able to study area PF due to its far lateral position, which yielded slices that were tangential to the pial surface. Areas PFG, PG, and Opt were in the convexity close to the lateral sulcus, while PFop and PGop were positioned more posteriorly, in the opercular region of IPC. Of all the five regions, area Opt was the one most similar to its analogue in the macaque, especially as revealed with SMI-32 staining. Namely, in both primate species area Opt showed a low density of large pyramidal neurons. Additionally, the apical dendrites of these neurons were sparse and vertically orientated, resembling columns. We also found area PG to be similar: both species exhibited cell body layers with a radial arrangement. On the other hand, Nissl staining revealed area PFG to be architectonically different between New and Old-World monkeys: PFG in the capuchin showed a comparatively higher cell density than in macaques, especially in layers II and IV. These results suggest that evolution may have enabled the functional specialization of these brain regions based on behavioral demands of upper limb use. The small differences in the IPC of the two primates may be linked to interspecies variability.

Hippocampal interneurons are direct targets for circulating glucocorticoids.

The hippocampus has become a significant target of stress research in recent years because of its role in cognitive functioning, neuropathology, and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Despite the pervasive impact of stress on psychiatric and neurological disease, many of the circuit- and cell-dependent mechanisms giving rise to the limbic regulation of the stress response remain unknown. Hippocampal excitatory neurons generally express high levels of glucocorticoid receptors (GRs) and are therefore positioned to respond directly to serum glucocorticoids. These neurons are, in turn, regulated by neighboring interneurons, subtypes of which have been shown to respond to stress exposure. However, GR expression among hippocampal interneurons is not well characterized. To determine whether key interneuron populations are direct targets for glucocorticoid action, we used two transgenic mouse lines to label parvalbumin-positive (PV+) and somatostatin-positive (SST+) interneurons. GR immunostaining of labeled interneurons was characterized within the dorsal and ventral dentate hilus, dentate cell body layer, and CA1 and CA3 stratum oriens and stratum pyramidale. While nearly all hippocampal SST+ interneurons expressed GR across all regions, GR labeling of PV+ interneurons showed considerable subregion variability. The percentage of PV+, GR+ cells was highest in the CA3 stratum pyramidale and lowest in the CA1 stratum oriens, with other regions showing intermediate levels of expression. Together, these findings indicate that, under baseline conditions, hippocampal SST+ interneurons are a ubiquitous glucocorticoid target, while only distinct populations of PV+ interneurons are direct targets. This anatomical diversity suggests functional differences in the regulation of stress-dependent hippocampal responses.

The synaptic inputs and thalamic projections of two classes of layer 6 corticothalamic neurons in primary somatosensory cortex of the mouse.

Although corticothalamic neurons (CThNs) represent the largest source of synaptic input to thalamic neurons, their role in regulating thalamocortical interactions remains incompletely understood. CThNs in sensory cortex have historically been divided into two types, those with cell bodies in Layer 6 (L6) that project back to primary sensory thalamic nuclei and those with cell bodies in Layer 5 (L5) that project to higher-order thalamic nuclei and subcortical structures. Recently, diversity among L6 CThNs has increasingly been appreciated. In the rodent somatosensory cortex, two major classes of L6 CThNs have been identified: one projecting to the ventral posterior medial nucleus (VPM-only L6 CThNs) and one projecting to both VPM and the posterior medial nucleus (VPM/POm L6 CThNs). Using rabies-based tracing methods in mice, we asked whether these L6 CThN populations integrate similar synaptic inputs. We found that both types of L6 CThNs received local input from somatosensory cortex and thalamic input from VPM and POm. However, VPM/POm L6 CThNs received significantly more input from a number of additional cortical areas, higher order thalamic nuclei, and subcortical structures. We also found that the two types of L6 CThNs target different functional regions within the thalamic reticular nucleus (TRN). Together, our results indicate that these two types of L6 CThNs represent distinct information streams in the somatosensory cortex and suggest that VPM-only L6 CThNs regulate, via their more restricted circuits, sensory responses related to a cortical column while VPM/POm L6 CThNs, which are integrated into more widespread POm-related circuits, relay contextual information.

3D Analysis of the Synaptic Organization in the Entorhinal Cortex in Alzheimer's Disease.

The entorhinal cortex (EC) is especially vulnerable in the early stages of Alzheimer’s disease (AD). In particular, cognitive deficits have been linked to alterations in the upper layers of EC. In the present report, we examined Layers II and III from eight human brain autopsies (four subjects with no recorded neurologic alterations and four AD cases). We used stereological methods to assess cortical atrophy of the EC and possible changes in the volume occupied by different cortical elements (neuronal and glial cell bodies; blood vessels; and neuropil). We performed 3D ultrastructural analyses of synapses using focused ion beam/scanning electron microscopy (FIB/SEM) to examine possible alterations related to AD. At the light microscope level, we found a significantly lower volume fraction occupied by neuronal bodies in Layer III and a higher volume fraction occupied by glial cell bodies in Layer II in AD cases. At the ultrastructural level, we observed that (1) there was a significantly lower synaptic density in both layers in AD cases; (2) synapses were larger and more complex in Layer II in AD cases; and (3) there was a greater proportion of small and simple synapses in Layer III in AD cases than in control individuals. These structural differences may play a role in the anatomic basis for the impairment of cognitive functions in AD.

Stimulation and Recording of the Hippocampus Using the Same Pt-Ir Coated Microelectrodes.

Same-electrode stimulation and recording with high spatial resolution, signal quality, and power efficiency is highly desirable in neuroscience and neural engineering. High spatial resolution and signal-to-noise ratio is necessary for obtaining unitary activities and delivering focal stimulations. Power efficiency is critical for battery-operated implantable neural interfaces. This study demonstrates the capability of recording single units as well as evoked potentials in response to a wide range of electrochemically safe stimulation pulses through high-resolution microelectrodes coated with co-deposition of Pt-Ir. It also compares signal-to-noise ratio, single unit activity, and power efficiencies between Pt-Ir coated and uncoated microelectrodes. To enable stimulation and recording with the same microelectrodes, microelectrode arrays were treated with electrodeposited platinum-iridium coating (EPIC) and tested in the CA1 cell body layer of rat hippocampi. The electrodes’ ability to (1) inject a large range of electrochemically reversable stimulation pulses to the tissue, and (2) record evoked potentials and single unit activities were quantitively assessed over an acute time period. Compared to uncoated electrodes, EPIC electrodes recorded signals with higher signal-to-noise ratios (coated: 9.77 ± 1.95 dB; uncoated: 1.95 ± 0.40 dB) and generated lower voltages (coated: 100 mV; uncoated: 650 mV) for a given stimulus (5 μA). The improved performance corresponded to lower energy consumptions and electrochemically safe stimulation above 5 μA (>0.38 mC/cm2), which enabled elicitation of field excitatory post synaptic potentials and population spikes. Spontaneous single unit activities were also modulated by varying stimulation intensities and monitored through the same electrodes. This work represents an example of stimulation and recording single unit activities from the same microelectrode, which provides a powerful tool for monitoring and manipulating neural circuits at the single neuron level.

In vivo large-scale analysis of Drosophila neuronal calcium traces by automated tracking of single somata

How does the concerted activity of neuronal populations shape behavior? Impediments to address this question are primarily due to critical experimental barriers. An integrated perspective on large scale neural information processing requires an in vivo approach that can combine the advantages of exhaustively observing all neurons dedicated to a given type of stimulus, and simultaneously achieve a resolution that is precise enough to capture individual neuron activity. Current experimental data from in vivo observations are either restricted to a small fraction of the total number of neurons, or are based on larger brain volumes but at a low spatial and temporal resolution. Consequently, fundamental questions as to how sensory information is represented on a population scale remain unanswered. In Drosophila melanogaster, the mushroom body (MB) represents an excellent model to analyze sensory coding and memory plasticity. In this work, we present an experimental setup coupled with a dedicated computational method that provides in vivo measurements of the activity of hundreds of densely packed somata uniformly spread in the MB. We exploit spinning-disk confocal 3D imaging over time of the whole MB cell body layer in vivo while it is exposed to olfactory stimulation. Importantly, to derive individual signal from densely packed somata, we have developed a fully automated image analysis procedure that takes advantage of the specificities of our data. After anisotropy correction, our approach operates a dedicated spot detection and registration over the entire time sequence to transform trajectories to identifiable clusters. This enabled us to discard spurious detections and reconstruct missing ones in a robust way. We demonstrate that this approach outperformed existing methods in this specific context and made possible high-throughput analysis of approximately 500 single somata uniformly spread over the MB in various conditions. Applying this approach, we find that learned experiences change the population code of odor representations in the MB. After long-term memory (LTM) formation, we quantified an increase in responsive somata count and a stable single neuron signal. We predict that this method, which should further enable studying the population pattern of neuronal activity, has the potential to uncover fine details of sensory processing and memory plasticity.

Acute in vivo testing of a conformal polymer microelectrode array for multi-region hippocampal recordings

Objective. The success of a cortical prosthetic device relies upon its ability to attain resolvable spikes from many neurons in particular neural networks over long periods of time. Traditionally, lifetimes of neural recordings are greatly limited by the body’s immune response against the foreign implant which causes neuronal death and glial scarring. This immune reaction is posited to be exacerbated by micromotion between the implant, which is often rigid, and the surrounding, soft brain tissue, and attenuates the quality of recordings over time. Approach. In an attempt to minimize the foreign body response to a penetrating neural array that records from multiple brain regions, Parylene C, a flexible, biocompatible polymer was used as the substrate material for a functional, proof-of-concept neural array with a reduced elastic modulus. This probe array was designed and fabricated to have 64 electrodes positioned to match the anatomy of the rat hippocampus and allow for simultaneous recordings between two cell-body layers of interest. A dissolvable brace was used for deep-brain penetration of the flexible array. Main results. Arrays were electrochemically characterized at the benchtop, and a novel insertion technique that restricts acute insertion injury enabled accurate target placement of four, bare, flexible arrays to greater than 4 mm deep into the rat brain. Arrays were tested acutely and in vivo recordings taken intra-operatively reveal spikes in both targeted regions of the hippocampus with spike amplitudes and noise levels similar to those recorded with microwires. Histological staining of a sham array implanted for one month reveals limited astrocytic scarring and neuronal death around the implant. Significance. This work represents one of the first examples of a penetrating polymer probe array that records from individual neurons in structures that lie deep within the brain.

Seizure liability assessments using the hippocampal tissue slice: Comparison of non-clinical species

IntroductionTraditionally, rat hippocampal tissue slice models are used as an in vitro electrophysiology assay to assess seizurogenic potential in early drug development despite non-clinical species-specific differences noted during in vivo seizure studies. MethodsHippocampal tissue slices were acutely isolated from rats, minipigs, dogs and nonhuman primates (NHP). Population spikes (PS) were evoked through stimulation of the CA3 Schaffer collateral pathway and recorded using in vitro electrophysiological techniques via an extracellular electrode placed within the CA1 stratum pyramidale cell body layer. ResultsHippocampal slices, across all species, displayed a concentration-dependent increase in PS area and number with the pro-convulsant pentylenetetrazol (PTZ; 0.1–10mM). Beagle dogs exhibited higher sensitivities to PTZ-induced changes in PS area and number compared to both rats and NHPs which presented nuanced differences in their responsiveness to PTZ modulation. Minipigs were comparatively resistant to PTZ-induced changes in both PS area and number. Rat and NHP hippocampal tissues were further characterized with the pro-convulsant agents 4-aminopyradine (4-AP; 1–100μM) and cefazolin (0.001–10mM). Rats possessed higher sensitivities to 4-AP- and cefazolin-induced changes to both PS area and number whereas NHP displayed greater modulation in PS duration. The anti-convulsant agents, diazepam (10–500μM) and lidocaine (1–500μM), were also tested on either rat and/or NHP tissue with both drugs repressing PS activation at high concentrations. DiscussionHippocampal tissue slices, across all species, possessed distinct sensitivities to pro- and anti-convulsant agents which may benefit the design of non-clinical seizure liability studies and their associated data interpretation.

Arrested Foveal Development in Preterm Eyes: Thickening of the Outer Nuclear Layer and Structural Redistribution Within the Fovea

The aim of this study was to define landmarks to better characterize foveal microstructure in normal subjects and in preterms with or without signs of immaturity, and to report on thickness changes of outer foveal layers following analysis of optical coherence tomography (OCT) B-scan images. Selected eyes from eight young adults with a history of prematurity (24-33 weeks of gestation) and five controls were imaged using conventional and directional OCT. Retinal layer thickness analysis was performed at selected temporal eccentricities defined by the individual distance between two landmarks for each case, the foveal center and the foveal rim. The use of a foveal center and foveal rim landmark transformation enabled comparisons of interindividual B-scans at corresponding landmark positions in both controls and preterms. We found a 20% shorter foveal center to foveal rim distance in preterms with an immature fovea than in controls. Reflectometric and manual segmentation measurements showed increased thickness of inner retinal layers and photoreceptor cell body and outer plexiform layers centrally, but no observable change of photoreceptor inner and outer segment thickness. Our landmark-based analysis of OCT images using reflectometry and manual segmentation provides complementary findings in comparisons of normal and immature foveal structures. We show a central thickness increase in the outer nuclear layer, outer plexiform layer, and postreceptor layers in preterms with signs of arrested foveal development. We found no indication of abnormal photoreceptor inner or outer segment development in preterms.

Redistribution within retinal layers of the central fovea in preterms with developmental arrest

PurposeThe aim of this study was to characterize thickness changes of foveal layers in preterms with or without signs of immaturity compared to control subjects through analysis of optical coherence tomography (OCT) B‐scan images.MethodsSelected eyes from 8 young adults with a history of prematurity (24‐33 weeks of gestation) and 5 controls were imaged using conventional and directional OCT. Retinal layer thickness analysis was performed at selected temporal eccentricities defined by the individual distance between two landmarks for each case, the foveal center (FC) and the foveal rim (R).ResultsThe use of an FC/R landmark transformation enabled comparisons of thickness profiles and area calculations from inter‐individual B‐scans at corresponding landmark positions in both controls and preterms. Reflectometric and manual segmentation measurements showed increased thickness of inner retinal layers (IRL) and photoreceptor cell body and axon/pedicle layers within the central fovea. Thickness and areal differences between controls and preterms with signs of developmental arrest showed a marked redistribution of IRL and the layers of photoreceptor cell bodies, axons and pedicles/synapses within the central fovea. This is in line with incomplete displacement of these layers from the foveal center. Since no changes of photoreceptor inner and outer segment thickness were observed the maturation of the photoreceptor segment layers seems to be within normal limits.ConclusionsOur landmark‐based analysis of OCT images using reflectometry and manual segmentation provides complementary findings in comparisons of normal and immature foveal structures. The thickness increase in the outer nuclear, Henle fiber, outer plexiform and post‐receptor layers in preterms with signs of arrested foveal development indicate a marked redistribution of cells.

Changes to the bladder epithelial barrier are associated with ketamine-induced cystitis.

The aim of the present study was to investigate the changes of the bladder epithelial barrier in the pathogenesis of ketamine-induced cystitis (KIC). A total of 60 female mice were randomly allocated into control and ketamine groups, which received daily intraperitoneal injections of saline and ketamine, respectively. Micturition behavior was recorded in 2-h intervals at the end of 4, 8 and 12 weeks, and bladders were harvested for subsequent analyses. Routine hematoxylin and eosin staining was performed on the bladders and histopathological changes were analyzed using light microscopy. The distribution of zonula occludens-1 (ZO-1) protein was determined by immunohistochemical analysis. The ultrastructure of umbrella cells was observed using a transmission electron microscope (TEM). Ketamine-addicted mice exhibited a significantly increased frequency of micturitions following 8 and 12 weeks of ketamine treatment (P<0.05 and P<0.01, respectively). Suburothelial congestion and infiltration of mononuclear cells was observed in ketamine-addicted mice following 8 and 12 weeks of treatment. Immunohistochemical examination demonstrated that there was an increased abnormal distribution of ZO-1 in the bladders of ketamine-treated mice compared with control mice. TEM analysis demonstrated that the surface of bladder urothelium became flattened, the tight junctions between umbrella cells became thinner and the endothelial cells exhibited cell body shrinkage, chromatin condensation and layer denudation in mice treated with ketamine. The present study indicated that the structural and functional changes to the bladder epithelial barrier caused by long-term use of ketamine may be key mechanisms in the development of KIC.

Distinct Localization of SNAP47 Protein in GABAergic and Glutamatergic Neurons in the Mouse and the Rat Hippocampus.

Synaptosomal-associated protein of 47 kDa (SNAP47) isoform is an atypical member of the SNAP family, which does not contribute directly to exocytosis and synaptic vesicle (SV) recycling. Initial characterization of SNAP47 revealed a widespread expression in nervous tissue, but little is known about its cellular and subcellular localization in hippocampal neurons. Therefore, in the present study we applied multiple-immunofluorescence labeling, immuno-electron microscopy and in situ hybridization (ISH) and analyzed the localization of SNAP47 in pre- and postsynaptic compartments of glutamatergic and GABAergic neurons in the mouse and rat hippocampus. While the immunofluorescence signal for SNAP47 showed a widespread distribution in both mouse and rat, the labeling pattern was complementary in the two species: in the mouse the immunolabeling was higher over the CA3 stratum radiatum, oriens and cell body layer. In contrast, in the rat the labeling was stronger over the CA1 neuropil and in the CA3 stratum lucidum. Furthermore, in the mouse high somatic labeling for SNAP47 was observed in GABAergic interneurons (INs). On the contrary, in the rat, while most INs were positive, they blended in with the high neuropil labeling. ISH confirmed the high expression of SNAP47 RNA in INs in the mouse. Co-staining for SNAP47 and pre- and postsynaptic markers in the rat revealed a strong co-localization postsynaptically with PSD95 in dendritic spines of pyramidal cells and, to a lesser extent, presynaptically, with ZnT3 and vesicular glutamate transporter 1 (VGLUT1) in glutamatergic terminals such as mossy fiber (MF) boutons. Ultrastructural analysis confirmed the pre- and postsynaptic localization at glutamatergic synapses. Furthermore, in the mouse hippocampus SNAP47 was found to be localized at low levels to dendritic shafts and axon terminals of putative INs forming symmetric synapses, indicating that this protein could be trafficked to both post- and presynaptic sites in both major cell types. These results reveal divergent localization of SNAP47 protein in mouse and rat hippocampus indicating species- and cell type-specific differences. SNAP47 is likely to be involved in unique fusion machinery which is distinct from the one involved in presynaptic neurotransmitter release. Nonetheless, our data suggest that SNAP47 may be involved not only postsynaptic, but also in presynaptic function.

Chronic multi-region recording from the rat hippocampus in vivo with a flexible Parylene-based multi-electrode array.

Neural activities of free-moving animals provide valuable insights into behavior, memory formation and cognitive function of the hippocampus. Unitary activities simultaneously recorded from multiple sub-regions of the hippocampus enable detailed study of hippocampal neural circuits, but require high fidelity recordings with high temporal and spatial resolution. In this work, we explored the possibility of using Parylene-C as the structural material for a penetrating, multi-electrode array designed to record from multiple sub-region of the rat hippocampus. A 64-channel Parylene-based flexible electrode array was designed and fabricated. The layout of the electrode array was arranged to conform to the shape of cell body layers of the rat hippocampus. An insertion technique of temporarily reduce the effective length of the probe with polyethylene glycol (PEG) was developed and tested in vivo. The multi-electrode array was implanted into a rat hippocampus for chronic experimentation and unitary activities were collected both during the implantation and after recovery while the animal ran freely in an open field. Unitary activities with an average signal to noise ratios (SNR) of 3 to 4 were recorded with the Parylene probe over the period of one month after implantation.

A flexible parylene probe for in vivo recordings from multiple subregions of the rat hippocampus.

The hippocampus is crucial to the formation of long-term memory and declarative memory. It is divided into three sub-fields the CA1, the CA3 and the DG. To understand the neuronal circuitry within the hippocampus and to study the role of the hippocampus in memory function requires the collection of neural activities from multiple subregions of the hippocampus simultaneously. Micro-wire electrode arrays are commonly used as an interface with neural systems. However, recording from multiple deep brain regions with curved anatomical structures such as the thin cell body layers of the hippocampus requires the micro-wires to be arranged into a highly accurate, complex layout that is difficult to fabricated manually. In this work, we designed and developed a flexible parylene-C based neural probe which can be easily micro-machined to the desired dimensions. Sixty-four electrical recording sites are micromachined on to 8 parylene shanks and spaced according to the distribution of hippocampal principal neurons in different hippocampus subregions. Together with our collaborators, we developed and optimized the implantation procedure of the flexible parylene probe and tested the insertion method both in brain tissue phantom and in vivo with a sham device. Immunohistochemistry (IHC) staining post-implantation of the sham probe was used to verify the location of the probe and to evaluate immune responses to the probe. Fully functional devices were fabricated and, in future studies, functional probes will be chronically implanted into the rat hippocampus, and neural activities will be recorded and compared with signals obtained with micro-wire arrays.