Nitrergic neurons of the forepaw representation in the rat somatosensory and motor cortices: A quantitative study.

Nitrergic neurons (NNs) are inhibitory neurons capable of releasing nitric oxide (NO) that are labeled with nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. The rat primary somatosensory (S1) and motor (M1) cortices are a favorable model to investigate NN populations by comparing their morphology, since these areas share the border of forepaw representation. The distribution of the Type I NN of the forepaw representation in the S1 and M1 cortices of the rat in different laminar compartments and the morphological parameters related to the cell body and dendritic arborization were measured and compared. We observed that the neuronal density in the S1 (130 NN/mm3 ) was higher than the neuronal density in the M1 (119 NN/mm3 ). Most NN neurons were multipolar (S1 with 58%; M1 with 69%), and a minority of the NN neurons were horizontal (S1 with 6%; M1 with 12%). NN found in S1 had a higher verticality index than NN found in M1, and no significant differences were observed for the other morphological parameters. We also demonstrated significant differences in most of the morphological parameters of the NN between different cortical compartments of S1 and M1. Our results indicate that the NN of the forepaw in S1 and M1 corresponds to a neuronal population, where the functionality is independent of the different types of sensory and motor processing. However, the morphological differences found between the cortical compartments of S1 and M1, as well as the higher density of NNs found in S1, indicate that the release of NO varies between the areas.

Cortical RORß is required for layer 4 transcriptional identity and barrel integrity.

Retinoic acid-related orphan receptor beta (RORß) is a transcription factor (TF) and marker of layer 4 (L4) neurons, which are distinctive both in transcriptional identity and the ability to form aggregates such as barrels in rodent somatosensory cortex. However, the relationship between transcriptional identity and L4 cytoarchitecture is largely unknown. We find RORß is required in the cortex for L4 aggregation into barrels and thalamocortical afferent (TCA) segregation. Interestingly, barrel organization also degrades with age in wildtype mice. Loss of RORß delays excitatory input and disrupts gene expression and chromatin accessibility, with down-regulation of L4 and up-regulation of L5 genes, suggesting a disruption in cellular specification. Expression and binding site accessibility change for many other TFs, including closure of neurodevelopmental TF binding sites and increased expression and binding capacity of activity-regulated TFs. Lastly, a putative target of RORß, Thsd7a, is down-regulated without RORß, and Thsd7a knock-out alone disrupts TCA organization in adult barrels.

Insights Into Spinal Dorsal Horn Circuit Function and Dysfunction Using Optical Approaches.

Somatosensation encompasses a variety of essential modalities including touch, pressure, proprioception, temperature, pain, and itch. These peripheral sensations are crucial for all types of behaviors, ranging from social interaction to danger avoidance. Somatosensory information is transmitted from primary afferent fibers in the periphery into the central nervous system via the dorsal horn of the spinal cord. The dorsal horn functions as an intermediary processing center for this information, comprising a complex network of excitatory and inhibitory interneurons as well as projection neurons that transmit the processed somatosensory information from the spinal cord to the brain. It is now known that there can be dysfunction within this spinal cord circuitry in pathological pain conditions and that these perturbations contribute to the development and maintenance of pathological pain. However, the complex and heterogeneous network of the spinal dorsal horn has hampered efforts to further elucidate its role in somatosensory processing. Emerging optical techniques promise to illuminate the underlying organization and function of the dorsal horn and provide insights into the role of spinal cord sensory processing in shaping the behavioral response to somatosensory input that we ultimately observe. This review article will focus on recent advances in optogenetics and fluorescence imaging techniques in the spinal cord, encompassing findings from both in vivo and in vitro preparations. We will also discuss the current limitations and difficulties of employing these techniques to interrogate the spinal cord and current practices and approaches to overcome these challenges.

jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths.

Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.

Remyelination alters the pattern of myelin in the cerebral cortex.

Destruction of oligodendrocytes and myelin sheaths in cortical gray matter profoundly alters neural activity and is associated with cognitive disability in multiple sclerosis (MS). Myelin can be restored by regenerating oligodendrocytes from resident progenitors; however, it is not known whether regeneration restores the complex myelination patterns in cortical circuits. Here, we performed time lapse in vivo two photon imaging in somatosensory cortex of adult mice to define the kinetics and specificity of myelin regeneration after acute oligodendrocyte ablation. These longitudinal studies revealed that the pattern of myelination in cortex changed dramatically after regeneration, as new oligodendrocytes were formed in different locations and new sheaths were often established along axon segments previously lacking myelin. Despite the dramatic increase in axonal territory available, oligodendrogenesis was persistently impaired in deeper cortical layers that experienced higher gliosis. Repeated reorganization of myelin patterns in MS may alter circuit function and contribute to cognitive decline.

Somatosensory corticospinal tract axons sprout within the cervical cord following a dorsal root/dorsal column spinal injury in the rat.

The corticospinal tract (CST) is the major descending pathway controlling voluntary hand function in primates, and though less dominant, it mediates voluntary paw movements in rats. As with primates, the CST in rats originates from multiple (albeit fewer) cortical sites, and functionally different motor and somatosensory subcomponents terminate in different regions of the spinal gray matter. We recently reported in monkeys that following a combined cervical dorsal root/dorsal column lesion (DRL/DCL), both motor and S1 CSTs sprout well beyond their normal terminal range. The S1 CST sprouting response is particularly dramatic, indicating an important, if poorly understood, somatosensory role in the recovery process. As rats are used extensively to model spinal cord injury, we asked if the S1 CST response is conserved in rodents. Rats were divided into sham controls, and two groups surviving post-lesion for ~6 and 10 weeks. A DRL/DCL was made to partially deafferent one paw. Behavioral testing showed a post-lesion deficit and recovery over several weeks. Three weeks prior to ending the experiment, S1 cortex was mapped electrophysiologically, for tracer injection placement to determine S1 CST termination patterns within the cord. Synaptogenesis was also assessed for labeled S1 CST terminals within the dorsal horn. Our findings show that the affected S1 CST sprouts well beyond its normal range in response to a DRL/DCL, much as it does in macaque monkeys. This, along with evidence for increased synaptogenesis post-lesion, indicates that CST terminal sprouting following a central sensory lesion, is a robust and conserved response.

Somatostatin receptors (SSTR1-5) on inhibitory interneurons in the barrel cortex.

Inhibitory interneurons in the cerebral cortex contain specific proteins or peptides characteristic for a certain interneuron subtype. In mice, three biochemical markers constitute non-overlapping interneuron populations, which account for 80-90% of all inhibitory cells. These interneurons express parvalbumin (PV), somatostatin (SST), or vasoactive intestinal peptide (VIP). SST is not only a marker of a specific interneuron subtype, but also an important neuropeptide that participates in numerous biochemical and signalling pathways in the brain via somatostatin receptors (SSTR1-5). In the nervous system, SST acts as a neuromodulator and neurotransmitter affecting, among others, memory, learning, and mood. In the sensory cortex, the co-localisation of GABA and SST is found in approximately 30% of interneurons. Considering the importance of interactions between inhibitory interneurons in cortical plasticity and the possible GABA and SST co-release, it seems important to investigate the localisation of different SSTRs on cortical interneurons. Here, we examined the distribution of SSTR1-5 on barrel cortex interneurons containing PV, SST, or VIP. Immunofluorescent staining using specific antibodies was performed on brain sections from transgenic mice that expressed red fluorescence in one specific interneuron subtype (PV-Ai14, SST-Ai14, and VIP-Ai14 mice). SSTRs expression on PV, SST, and VIP interneurons varied among the cortical layers and we found two patterns of SSTRs distribution in L4 of barrel cortex. We also demonstrated that, in contrast to other interneurons, PV cells did not express SSTR2, but expressed other SSTRs. SST interneurons, which were not found to make chemical synapses among themselves, expressed all five SSTR subtypes.

Development and sensory experience dependent regulation of microglia in barrel cortex.

The barrel cortex is within the primary somatosensory cortex of the rodent, and processes signals from the vibrissae. Much focus has been devoted to the function of neurons, more recently, the role of glial cells in the processing of sensory input has gained increasing interest. Microglia are the principal immune cells of the nervous system that survey and regulate the cellular constituents of the dynamic nervous system. We investigated the normal and disrupted development of microglia in barrel cortex by chronically depriving sensory signals via whisker trimming for the animals' first postnatal month. Using immunohistochemistry to label microglia, we performed morphological reconstructions as well as densitometry analyses as a function of developmental age and sensory experience. Findings suggest that both developmental age and sensory experience has profound impact on microglia morphology. Following chronic sensory deprivation, microglia undergo a morphological transition from a monitoring or resting state to an altered morphological state, by exhibiting expanded cell body size and retracted processes. Sensory restoration via whisker regrowth returns these morphological alterations back to age-matched control values. Our results indicate that microglia may be recruited to participate in the modulation of neuronal structural remodeling during developmental critical periods and in response to alteration in sensory input.

Architectonic features and relative locations of primary sensory and related areas of neocortex in mouse lemurs.

Mouse lemurs are the smallest of the living primates, and are members of the understudied radiation of strepsirrhine lemurs of Madagascar. They are thought to closely resemble the ancestral primates that gave rise to present day primates. Here we have used multiple histological and immunochemical methods to identify and characterize sensory areas of neocortex in four brains of adult lemurs obtained from a licensed breeding colony. We describe the laminar features for the primary visual area (V1), the secondary visual area (V2), the middle temporal visual area (MT) and area prostriata, somatosensory areas S1(3b), 3a, and area 1, the primary motor cortex (M1), and the primary auditory cortex (A1). V1 has "blobs" with "nonblob" surrounds, providing further evidence that this type of modular organization might have evolved early in the primate lineage to be retained in all extant primates. The laminar organization of V1 further supports the view that sublayers of layer 3 of primates have been commonly misidentified as sublayers of layer 4. S1 (area 3b) is proportionately wider than the elongated area observed in anthropoid primates, and has disruptions that may distinguish representations of the hand, face, teeth, and tongue. Primary auditory cortex is located in the upper temporal cortex and may include a rostral area, R, in addition to A1. The resulting architectonic maps of cortical areas in mouse lemurs can usefully guide future studies of cortical connectivity and function.

Gap Junctions Interconnect Different Subtypes of Parvalbumin-Positive Interneurons in Barrels and Septa with Connectivity Unique to Each Subtype.

Parvalbumin (PV)-positive interneurons form dendritic gap junctions with one another, but the connectivity among gap junction-coupled dendrites remains uninvestigated in most neocortical areas. We visualized gap junctions in layer 4 of the mouse barrel cortex and examined their structural details. PV neurons were divided into 4 types based on the location of soma and dendrites within or outside barrels. Type 1 neurons that had soma and all dendrites inside a barrel, considered most specific to single vibrissa-derived signals, unexpectedly formed gap junctions only with other types but never with each other. Type 2 neurons inside a barrel elongated dendrites outward, forming gap junctions within a column that contained the home barrel. Type 3 neurons located outside barrels established connections with all types including Type 4 neurons that were confined inside the inter-barrel septa. The majority (33/38, 86.8%) of dendritic gap junctions were within 75 µm from at least 1 of 2 paired somata. All types received vesicular glutamate transporter 2-positive axon terminals preferentially on somata and proximal dendrites, indicating the involvement of all types in thalamocortical feedforward regulation in which proximal gap junctions may also participate. These structural organizations provide a new morphological basis for regulatory mechanisms in barrel cortex.

Inhibitory Network Bistability Explains Increased Interneuronal Activity Prior to Seizure Onset.

Recent experimental literature has revealed that GABAergic interneurons exhibit increased activity prior to seizure onset, alongside additional evidence that such activity is synchronous and may arise abruptly. These findings have led some to hypothesize that this interneuronal activity may serve a causal role in driving the sudden change in brain activity that heralds seizure onset. However, the mechanisms predisposing an inhibitory network toward increased activity, specifically prior to ictogenesis, without a permanent change to inputs to the system remain unknown. We address this question by comparing simulated inhibitory networks containing control interneurons and networks containing hyperexcitable interneurons modeled to mimic treatment with 4-Aminopyridine (4-AP), an agent commonly used to model seizures in vivo and in vitro. Our in silico study demonstrates that model inhibitory networks with 4-AP interneurons are more prone than their control counterparts to exist in a bistable state in which asynchronously firing networks can abruptly transition into synchrony driven by a brief perturbation. This transition into synchrony brings about a corresponding increase in overall firing rate. We further show that perturbations driving this transition could arise in vivo from background excitatory synaptic activity in the cortex. Thus, we propose that bistability explains the increase in interneuron activity observed experimentally prior to seizure via a transition from incoherent to coherent dynamics. Moreover, bistability explains why inhibitory networks containing hyperexcitable interneurons are more vulnerable to this change in dynamics, and how such networks can undergo a transition without a permanent change in the drive. We note that while our comparisons are between networks of control and ictogenic neurons, the conclusions drawn specifically relate to the unusual dynamics that arise prior to seizure, and not seizure onset itself. However, providing a mechanistic explanation for this phenomenon specifically in a pro-ictogenic setting generates experimentally testable hypotheses regarding the role of inhibitory neurons in pre-ictal neural dynamics, and motivates further computational research into mechanisms underlying a newly hypothesized multi-step pathway to seizure initiated by inhibition.

Membrane Potential Correlates of Network Decorrelation and Improved SNR by Cholinergic Activation in the Somatosensory Cortex.

The nucleus basalis (NB) projects cholinergic axons to the cortex, where they play a major role in arousal, attention, and learning. Cholinergic inputs shift cortical dynamics from synchronous to asynchronous and improve the signal-to-noise ratio (SNR) of sensory responses. However, the underlying mechanisms of these changes remain unclear. Using simultaneous extracellular and whole-cell patch recordings in layer 4 of the mouse barrel cortex, we show that electrical or optogenetic activation of the cholinergic system has a differential effect on ongoing and sensory evoked activities. Cholinergic activation profoundly reduced the large spontaneous fluctuations in membrane potential and decorrelated ongoing activity. However, NB stimulation had no effect on the response to whisker stimulation or on signal correlations. These effects of cholinergic activation provide a unified explanation for the increased SNR of sensory response and for the reduction in noise correlations and explain the shift into the desynchronized cortical state, which are the hallmarks of arousal and attention.SIGNIFICANCE STATEMENT Attention increases the signal-to-noise ratio (SNR) of cortical sensory response, which may reflect either reduction in background firing rate or increased sensory response. Extracellular recordings showed that attention also reduces the correlation in network activity. These effects are partially mediated by cholinergic axons from the nucleus basalis projecting to the entire cortex. To reveal the cellular and synaptic correlates of these cholinergic effects, we performed simultaneous intracellular and LFP recordings in the somatosensory cortex. Global or local cholinergic activation increased the SNR of sensory response mainly by reducing the rate and amplitude of background synaptic activity and also reduced network correlations. Therefore, coding of sensory information is enhanced by the cholinergic system mainly due to a reduction in spontaneous activity.

Site Specificity of Changes in Cortical Oxyhaemoglobin Concentration Induced by Water Immersion.

Our previous studies have shown that water immersion (WI) changes sensorimotor processing and cortical excitability in the sensorimotor regions of the brain. The present study examined the site specificity of the brain activation during WI using functional near infrared spectroscopy (fNIRS). Cortical oxyhaemoglobin (O2Hb) levels in the anterior and posterior parts of the supplementary motor area (pre-SMA and SMA), primary motor cortex (M1), primary somatosensory cortex (S1), and posterior parietal cortex (PPC) were recorded using fNIRS (OMM-3000; Shimadzu Co.) before, during, and after WI in nine healthy participants. The cortical O2Hb levels in SMA, M1, S1, and PPC significantly increased during the WI and increased gradually along with the filling of the WI tank. These changes were not seen in the pre-SMA. The results show that WI-induced increases in cortical O2Hb levels are at least somewhat site specific: there was little brain activation in response to somatosensory input in the pre-SMA, but robust activation in other areas.

Planar implantable sensor for in vivo measurement of cellular oxygen metabolism in brain tissue.

BACKGROUND: Brain imaging methods are continually improving. Imaging of the cerebral cortex is widely used in both animal experiments and charting human brain function in health and disease. Among the animal models, the rodent cerebral cortex has been widely used because of patterned neural representation of the whiskers on the snout and relative ease of activating cortical tissue with whisker stimulation. NEW METHOD: We tested a new planar solid-state oxygen sensor comprising a polymeric film with a phosphorescent oxygen-sensitive coating on the working side, to monitor dynamics of oxygen metabolism in the cerebral cortex following sensory stimulation. RESULTS: Sensory stimulation led to changes in oxygenation and deoxygenation processes of activated areas in the barrel cortex. We demonstrate the possibility of dynamic mapping of relative changes in oxygenation in live mouse brain tissue with such a sensor. COMPARISON WITH EXISTING METHOD: Oxygenation-based functional magnetic resonance imaging (fMRI) is very effective method for functional brain mapping but have high costs and limited spatial resolution. Optical imaging of intrinsic signal (IOS) does not provide the required sensitivity, and voltage-sensitive dye optical imaging (VSDi) has limited applicability due to significant toxicity of the voltage-sensitive dye. Our planar solid-state oxygen sensor imaging approach circumvents these limitations, providing a simple optical contrast agent with low toxicity and rapid application. CONCLUSIONS: The planar solid-state oxygen sensor described here can be used as a tool in visualization and real-time analysis of sensory-evoked neural activity in vivo. Further, this approach allows visualization of local neural activity with high temporal and spatial resolution.

Coordinated Plasticity between Barrel Cortical Glutamatergic and GABAergic Neurons during Associative Memory.

Neural plasticity is associated with memory formation. The coordinated refinement and interaction between cortical glutamatergic and GABAergic neurons remain elusive in associative memory, which we examine in a mouse model of associative learning. In the mice that show odorant-induced whisker motion after pairing whisker and odor stimulations, the barrel cortical glutamatergic and GABAergic neurons are recruited to encode the newly learnt odor signal alongside the innate whisker signal. These glutamatergic neurons are functionally upregulated, and GABAergic neurons are refined in a homeostatic manner. The mutual innervations between these glutamatergic and GABAergic neurons are upregulated. The analyses by high throughput sequencing show that certain microRNAs related to regulating synapses and neurons are involved in this cross-modal reflex. Thus, the coactivation of the sensory cortices through epigenetic processes recruits their glutamatergic and GABAergic neurons to be the associative memory cells as well as drive their coordinated refinements toward the optimal state for the storage of the associated signals.

Assessing Susceptibility to Epilepsy in Three Rat Strains Using Brain Metabolic Profiling Based on HRMAS NMR Spectroscopy and Chemometrics.

The possibility that a metabolomic approach can inform about the pathophysiology of a given form of epilepsy was addressed. Using chemometric analyses of HRMAS NMR data, we compared several brain structures in three rat strains with different susceptibilities to absence epilepsy: Genetic Absence Epilepsy Rats from Strasbourg (GAERS), Non Epileptic Control rats (NEC), and Wistar rats. Two ages were investigated: 14 days postnatal (P14) before the onset of seizures and 5 month old adults with fully developed seizures (Adults). The relative concentrations of 19 metabolites were assessed using (1)H HRMAS NMR experiments. Univariate and multivariate analyses including multiblock models were used to identify the most discriminant metabolites. A strain-dependent evolution of glutamate, glutamine, scyllo-inositol, alanine, and glutathione was highlighted during cerebral maturation. In Adults, data from somatosensory and motor cortices allowed discrimination between GAERS and NEC rats with higher levels of scyllo-inositol, taurine, and phosphoethanolamine in NEC. This epileptic metabolic phenotype was in accordance with current pathophysiological hypothesis of absence epilepsy (i.e., seizure-generating and control networks) and putative resistance of NEC rats and was observed before seizure onset. This methodology could be very efficient in a clinical context.

Altered glutamate/GABA equilibrium in aged mice cortex influences cortical plasticity.

Age-related molecular changes in the synapse can cause plasticity decline. We found an impairment of experience-dependent cortical plasticity is induced by short lasting sensory conditioning in aged mice. However, extending the training procedure from 3 to 7 days triggered plasticity in the aged cortex of the same range as in young mice. Additionally, GABAergic markers (GABA, GAD67, VGAT) in young and aged groups that showed the plastic changes were upregulated. This effect was absent in the aged group with impaired plasticity, while the expression of Vglut1 increased in all trained groups. This may reflect the inefficiency of inhibitory mechanisms in the aging brain used to control increased excitation after training and to shape proper signal to noise ratio, which is essential for appropriate stimuli processing. HPLC analysis showed that the glutamate/GABA ratio was significantly reduced in aged animals due to a significant decrease in glutamate level. We also observed a decreased expression of several presynaptic markers involved in excitatory (vesicular glutamate transporter-vglut2) and inhibitory (glutamic acid decarboxylase-GAD67, vesicular GABA transporter VGAT) transmission in the aged barrel cortex. These changes may weaken the plasticity potential of neurons and impede the experience-dependent reorganization of cortical connections. We suggest that the imbalance toward inhibition resulting from a decrease of glutamate content in the aging cerebral cortex, together with GABAergic system ineffectiveness in upregulating GABA level after sensory training, contributes to the impairment of learning-dependent cortical plasticity.

A high-throughput semi-automated preparation for filtered synaptoneurosomes.

BACKGROUND: Synaptoneurosomes have become an important tool for studying synaptic proteins. The filtered synaptoneurosomes preparation originally developed by Hollingsworth et al. (1985) is widely used and is an easy method to prepare synaptoneurosomes. The hand processing steps in that preparation, however, are labor intensive and have become a bottleneck for current proteomic studies using synaptoneurosomes. For this reason, we developed new steps for tissue homogenization and filtration that transform the preparation of synaptoneurosomes to a high-throughput, semi-automated process. NEW METHOD: We implemented a standardized protocol with easy to follow steps for homogenizing multiple samples simultaneously using a FastPrep tissue homogenizer (MP Biomedicals, LLC) and then filtering all of the samples in centrifugal filter units (EMD Millipore, Corp). RESULTS AND COMPARISON WITH EXISTING METHODS: The new steps dramatically reduce the time to prepare synaptoneurosomes from hours to minutes, increase sample recovery, and nearly double enrichment for synaptic proteins. These steps are also compatible with biosafety requirements for working with pathogen infected brain tissue. CONCLUSIONS: The new high-throughput semi-automated steps to prepare synaptoneurosomes are timely technical advances for studies of low abundance synaptic proteins in valuable tissue samples.

Synaptic molecular imaging in spared and deprived columns of mouse barrel cortex with array tomography.

A major question in neuroscience is how diverse subsets of synaptic connections in neural circuits are affected by experience dependent plasticity to form the basis for behavioral learning and memory. Differences in protein expression patterns at individual synapses could constitute a key to understanding both synaptic diversity and the effects of plasticity at different synapse populations. Our approach to this question leverages the immunohistochemical multiplexing capability of array tomography (ATomo) and the columnar organization of mouse barrel cortex to create a dataset comprising high resolution volumetric images of spared and deprived cortical whisker barrels stained for over a dozen synaptic molecules each. These dataset has been made available through the Open Connectome Project for interactive online viewing, and may also be downloaded for offline analysis using web, Matlab, and other interfaces.