Immune receptors and aging brain.

Aging brings about a myriad of degenerative processes throughout the body. A decrease in cognitive abilities is one of the hallmark phenotypes of aging, underpinned by neuroinflammation and neurodegeneration occurring in the brain. This review focuses on the role of different immune receptors expressed in cells of the central and peripheral nervous systems. We will discuss how immune receptors in the brain act as sentinels and effectors of the age-dependent shift in ligand composition. Within this 'old-age-ligand soup,' some immune receptors contribute directly to excessive synaptic weakening from within the neuronal compartment, while others amplify the damaging inflammatory environment in the brain. Ultimately, chronic inflammation sets up a positive feedback loop that increases the impact of immune ligand-receptor interactions in the brain, leading to permanent synaptic and neuronal loss.

The nonclassical MHC class I Qa-1 expressed in layer 6 neurons regulates activity-dependent plasticity via microglial CD94/NKG2 in the cortex.

During developmental critical periods, circuits are sculpted by a process of activity-dependent competition. The molecular machinery involved in regulating the complex process of responding to different levels of activity is now beginning to be identified. Here, we show that the nonclassical major histocompatibility class I (MHCI) molecule Qa-1 is expressed in the healthy brain in layer 6 corticothalamic neurons. In the visual cortex, Qa-1 expression begins during the critical period for ocular dominance (OD) plasticity and is regulated by neuronal activity, suggesting a role in regulating activity-dependent competition. Indeed, in mice lacking Qa-1, OD plasticity is perturbed. Moreover, signaling through CD94/NKG2, a known cognate Qa-1 heterodimeric receptor in the immune system, is implicated: selectively targeting this interaction phenocopies the plasticity perturbation observed in Qa-1 knockouts. In the cortex, CD94/NKG2 is expressed by microglial cells, which undergo activity-dependent changes in their morphology in a Qa-1­dependent manner. Our study thus reveals a neuron­microglial interaction dependent upon a nonclassical MHCI molecule expressed in L6 neurons, which regulates plasticity in the visual cortex. These results also point to an unexpected function for the Qa-1/HLA-E (ligand) and CD94/NKG2 (receptor) interaction in the nervous system, in addition to that described in the immune system.

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice.

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol's critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

Activity-dependent modulation of hippocampal synaptic plasticity via PirB and endocannabinoids.

The threshold for Hebbian synaptic plasticity in the CNS is modulated by prior synaptic activity. At adult CA3-CA1 synapses, endocannabinoids play a role in this process, but how activity engages and maintains this retrograde signaling system is not well understood. Here we show that conditional deletion of Paired Immunoglobulin-like receptor B (PirB) from pyramidal neurons in adult mouse hippocampus results in deficient LTD at CA3-CA1 synapses over a range of stimulation frequencies, accompanied by an increase in LTP. This finding can be fully explained by the disengagement of retrograde endocannabinoid signaling selectively at excitatory synapses. In the absence of PirB, the NMDAR-dependent regulation of endocannabinoid signaling is lost, while CB1R-dependent and group I mGluR-dependent regulation are intact. Moreover, mEPSC frequency in mutant CA1 pyramidal cells is elevated, consistent with a higher density of excitatory synapses and altered synapse pruning. Mice lacking PirB also perform better than WT in learning and memory tasks. These observations suggest that PirB is an integral part of an NMDA receptor-mediated synaptic mechanism that maintains bidirectional Hebbian plasticity and learning via activity-dependent endocannabinoid signaling.

Automated dendritic spine detection using convolutional neural networks on maximum intensity projected microscopic volumes.

BACKGROUND: Dendritic spines are structural correlates of excitatory synapses in the brain. Their density and structure are shaped by experience, pointing to their role in memory encoding. Dendritic spine imaging, followed by manual analysis, is a primary way to study spines. However, an approach that analyses dendritic spines images in an automated and unbiased manner is needed to fully capture how spines change with normal experience, as well as in disease. NEW METHOD: We propose an approach based on fully convolutional neural networks (FCNs) to detect dendritic spines in two-dimensional maximum-intensity projected images from confocal fluorescent micrographs. We experiment on both fractionally strided convolution and efficient sub-pixel convolutions. Dendritic spines far from the dendritic shaft are pruned by extraction of the shaft to reduce false positives. Performance of the proposed method is evaluated by comparing predicted spine positions to those manually marked by experts. RESULTS: The averaged distance between predicted and manually annotated spines is 2.81 ± 2.63 pixels (0.082 ± 0.076 microns) and 2.87 ± 2.33 pixels (0.084 ± 0.068 microns) based on two different experts. FCN-based detection achieves F scores > 0.80 for both sets of expert annotations. COMPARISON WITH EXISTING METHODS: Our method significantly outperforms two well-known software, NeuronStudio and Neurolucida (p-value < 0.02). CONCLUSIONS: FCN architectures used in this work allow for automated dendritic spine detection. Superior outcomes are possible even with small training data-sets. The proposed method may generalize to other datasets on larger scales.

Cell-Autonomous Regulation of Dendritic Spine Density by PirB.

Synapse density on cortical pyramidal neurons is modulated by experience. This process is highest during developmental critical periods, when mechanisms of synaptic plasticity are fully engaged. In mouse visual cortex, the critical period for ocular dominance (OD) plasticity coincides with the developmental pruning of synapses. At this time, mice lacking paired Ig-like receptor B (PirB) have excess numbers of dendritic spines on L5 neurons; these spines persist and are thought to underlie the juvenile-like OD plasticity observed in adulthood. Here we examine whether PirB is required specifically in excitatory neurons to exert its effect on dendritic spine and synapse density during the critical period. In mice with a conditional allele of PirB (PirBfl/fl), PirB was deleted only from L2/3 cortical pyramidal neurons in vivo by timed in utero electroporation of Cre recombinase. Sparse mosaic expression of Cre produced neurons lacking PirB in a sea of wild-type neurons and glia. These neurons had significantly elevated dendritic spine density, as well as increased frequency of miniature EPSCs, suggesting that they receive a greater number of synaptic inputs relative to Cre- neighbors. The effect of cell-specific PirB deletion on dendritic spine density was not accompanied by changes in dendritic branching complexity or axonal bouton density. Together, results imply a neuron-specific, cell-autonomous action of PirB on synaptic density in L2/3 pyramidal cells of visual cortex. Moreover, they are consistent with the idea that PirB functions normally to corepress spine density and synaptic plasticity, thereby maintaining headroom for cells to encode ongoing experience-dependent structural change throughout life.

Developmental Sculpting of Intracortical Circuits by MHC Class I H2-Db and H2-Kb.

Synapse pruning is an activity-regulated process needed for proper circuit sculpting in the developing brain. Major histocompatibility class I (MHCI) molecules are regulated by activity, but little is known about their role in the development of connectivity in cortex. Here we show that protein for 2 MHCI molecules H2-Kb and H2-Db is associated with synapses in the visual cortex. Pyramidal neurons in mice lacking H2-Kb and H2-Db (KbDb KO) have more extensive cortical connectivity than normal. Modified rabies virus tracing was used to monitor the extent of pyramidal cell connectivity: Horizontal connectivity is greater in the visual cortex of KbDb KO mice. Basal dendrites of L2/3 pyramids, where many horizontal connections terminate, are more highly branched and have elevated spine density in the KO. Furthermore, the density of axonal boutons is elevated within L2/3 of mutant mice. These increases are accompanied by elevated miniature excitatory postsynaptic current frequency, consistent with an increase in functional synapses. This functional and anatomical increase in intracortical connectivity is also associated with enhanced ocular dominance plasticity that persists into adulthood. Thus, these MHCI proteins regulate sculpting of local cortical circuits and in their absence, the excess connectivity can function as a substrate for cortical plasticity throughout life.

Blocking PirB up-regulates spines and functional synapses to unlock visual cortical plasticity and facilitate recovery from amblyopia.

During critical periods of development, the brain easily changes in response to environmental stimuli, but this neural plasticity declines by adulthood. By acutely disrupting paired immunoglobulin-like receptor B (PirB) function at specific ages, we show that PirB actively represses neural plasticity throughout life. We disrupted PirB function either by genetically introducing a conditional PirB allele into mice or by minipump infusion of a soluble PirB ectodomain (sPirB) into mouse visual cortex. We found that neural plasticity, as measured by depriving mice of vision in one eye and testing ocular dominance, was enhanced by this treatment both during the critical period and when PirB function was disrupted in adulthood. Acute blockade of PirB triggered the formation of new functional synapses, as indicated by increases in miniature excitatory postsynaptic current (mEPSC) frequency and spine density on dendrites of layer 5 pyramidal neurons. In addition, recovery from amblyopia--the decline in visual acuity and spine density resulting from long-term monocular deprivation--was possible after a 1-week infusion of sPirB after the deprivation period. Thus, neural plasticity in adult visual cortex is actively repressed and can be enhanced by blocking PirB function.

PirB regulates a structural substrate for cortical plasticity.

Experience-driven circuit changes underlie learning and memory. Monocular deprivation (MD) engages synaptic mechanisms of ocular dominance (OD) plasticity and generates robust increases in dendritic spine density on L5 pyramidal neurons. Here we show that the paired immunoglobulin-like receptor B (PirB) negatively regulates spine density, as well as the threshold for adult OD plasticity. In PirB(-/-) mice, spine density and stability are significantly greater than WT, associated with higher-frequency miniature synaptic currents, larger long-term potentiation, and deficient long-term depression. Although MD generates the expected increase in spine density in WT, in PirB(-/-) this increase is occluded. In adult PirB(-/-), OD plasticity is larger and more rapid than in WT, consistent with the maintenance of elevated spine density. Thus, PirB normally regulates spine and excitatory synapse density and consequently the threshold for new learning throughout life.

Human LilrB2 is a ß-amyloid receptor and its murine homolog PirB regulates synaptic plasticity in an Alzheimer's model.

Soluble ß-amyloid (Aß) oligomers impair synaptic plasticity and cause synaptic loss associated with Alzheimer's disease (AD). We report that murine PirB (paired immunoglobulin-like receptor B) and its human ortholog LilrB2 (leukocyte immunoglobulin-like receptor B2), present in human brain, are receptors for Aß oligomers, with nanomolar affinity. The first two extracellular immunoglobulin (Ig) domains of PirB and LilrB2 mediate this interaction, leading to enhanced cofilin signaling, also seen in human AD brains. In mice, the deleterious effect of Aß oligomers on hippocampal long-term potentiation required PirB, and in a transgenic model of AD, PirB not only contributed to memory deficits present in adult mice, but also mediated loss of synaptic plasticity in juvenile visual cortex. These findings imply that LilrB2 contributes to human AD neuropathology and suggest therapeutic uses of blocking LilrB2 function.

Neuroprotection from stroke in the absence of MHCI or PirB.

Recovery from stroke engages mechanisms of neural plasticity. Here we examine a role for MHC class I (MHCI) H2-Kb and H2-Db, as well as PirB receptor. These molecules restrict synaptic plasticity and motor learning in the healthy brain. Stroke elevates neuronal expression not only of H2-Kb and H2-Db, but also of PirB and downstream signaling. KbDb knockout (KO) or PirB KO mice have smaller infarcts and enhanced motor recovery. KO hippocampal organotypic slices, which lack an intact peripheral immune response, have less cell death after in vitro ischemia. In PirB KO mice, corticospinal projections from the motor cortex are enhanced, and the reactive astrocytic response is dampened after MCAO. Thus, molecules that function in the immune system act not only to limit synaptic plasticity in healthy neurons, but also to exacerbate brain injury after ischemia. These results suggest therapies for stroke by targeting MHCI and PirB.

Phosphorylation state of the Na+-K+-Cl- cotransporter (NKCC1) in the gills of Atlantic killifish (Fundulus heteroclitus) during acclimation to water of varying salinity.

Euryhaline teleosts such as Atlantic killifish (Fundulus heteroclitus) are able to acclimate to changing environmental salinity by tightly regulating NaCl absorption and secretion across their gills. Many studies have examined the mechanisms responsible for long-term (days) salinity acclimation; however, much remains unknown about the mechanisms of acute (hours) salinity acclimation. In this study, we tested the hypotheses that phosphorylation of the Na(+)-K(+)-Cl(-) cotransporter (NKCC1) located in the basolateral membrane of the gill plays a role in acute salinity acclimation and that changes in NKCC1 phosphorylation are mediated by a cAMP-protein kinase A (cAMP-PKA) pathway. Using a phospho-specific antibody, we determined the time course of changes in total and phosphorylated NKCC1 protein during acclimation to water of various salinities. Long-term (>or=14 days) acclimation of killifish to seawater (SW) and 2x SW resulted in 4- to 6-fold and 5- to 8-fold increases, respectively, in total gill NKCC1 protein relative to fish maintained in freshwater (FW). NKCC1 was found to be between 20% and 70% activated in fish, with lower average activation in fish acclimated to SW and 2x SW compared with FW fish. Increases and decreases in the fractional level of NKCC1 phosphorylation were seen within 1 h of transfer of fish to water of higher and lower salinity, respectively, consistent with a regulatory role of phosphorylation prior to an increase in the biosynthesis of NKCC1; large changes in protein expression of NKCC1 were observed over periods of hours to days. We found that NKCC1 phosphorylation is acutely regulated in the killifish gill in response to changing environmental salinity and that phosphorylation in excised gills increases in response to forskolin stimulation of the cAMP-PKA pathway. The role of phosphorylation is further underscored by the observation that mRNA expression of sterile 20 (Ste20)-related proline-alanine-rich kinase (SPAK) changes with salinity acclimation, being 2.7-fold greater in SW-acclimated killifish relative to FW fish. Overall, these results demonstrate an important role of NKCC1 phosphorylation in the gill of Atlantic killifish during acute salinity acclimation.

Wide-field and two-photon imaging of brain activity with voltage- and calcium-sensitive dyes.

This chapter presents three examples of imaging brain activity with voltage- or calcium-sensitive dyes. Because experimental measurements are limited by low sensitivity, the chapter then discusses the methodological aspects that are critical for optimal signal-to-noise ratio. Two of the examples use wide-field (1-photon) imaging and the third uses two-photon scanning microscopy. These methods have relatively high temporal resolution ranging from 10 to 10,000 Hz. The three examples are the following: (1) Internally injected voltage-sensitive dye can be used to monitor membrane potential in the dendrites of invertebrate and vertebrate neurons in in vitro preparations. These experiments are directed at understanding how individual neurons convert the complex input synaptic activity into the output spike train. (2) Recently developed methods for staining many individual cells in the mammalian brain with calcium-sensitive dyes together with two-photon microscopy made it possible to follow the spike activity of many neurons simultaneously while in vivo preparations are responding to stimulation. (3) Calcium-sensitive dyes that are internalized into olfactory receptor neurons in the nose will, after several days, be transported to the nerve terminals of these cells in the olfactory bulb glomeruli. There, the population signals can be used as a measure of the input from the nose to the bulb. Three kinds of noise in measuring light intensity are discussed: (1) Shot noise from the random emission of photons from the preparation. (2) Extraneous (technical) noise from external sources. (3) Noise that occurs in the absence of light, the dark noise. In addition, we briefly discuss the light sources, the optics, and the detectors and cameras. The commonly used organic voltage and ion sensitive dyes stain all of the cell types in the preparation indiscriminately. A major effort is underway to find methods for staining individual cell types in the brain selectively. Most of these efforts center around fluorescent protein activity sensors because transgenic methods can be used to express them in individual cell types.

Functional structure of the mitral cell dendritic tuft in the rat olfactory bulb.

The input-output transform performed by mitral cells, the principal projection neurons of the olfactory bulb, is one of the key factors in understanding olfaction. We used combined calcium and voltage imaging from the same neuron and computer modeling to investigate signal processing in the mitral cells, focusing on the glomerular dendritic tuft. The main finding was that the dendritic tuft functions as a single electrical compartment for subthreshold signals within the range of amplitudes detectable by voltage-sensitive dye recording. These evoked EPSPs had uniform characteristics throughout the glomerular tuft. The Ca(2+) transients associated with spatially uniform subthreshold synaptic potentials were comparable but not equal in amplitude in all regions. The average range of normalized amplitudes of the EPSP-driven Ca(2+) signals from different locations on dendritic branches in the glomerular tuft was relatively narrow and appeared to be independent of the dendritic surface-to-volume ratio. The computer simulations constrained by the imaging data indicated that a synchronized activation of approximately 100 synapses randomly distributed on tuft branches was sufficient to generate spatially homogenous EPSPs. This number of activated synapses is consistent with the data from anatomical studies. Furthermore, voltage attenuation of the EPSP along the primary dendrite at physiological temperature was weak compared with other cell types. In the model, weak attenuation of the EPSP along the primary dendrite could be accounted for by passive electrical properties of the mitral cell.

Dendritic signals from rat hippocampal CA1 pyramidal neurons during coincident pre- and post-synaptic activity: a combined voltage- and calcium-imaging study.

The non-linear and spatially inhomogeneous interactions of dendritic membrane potential signals that represent the first step in the induction of activity-dependent long-term synaptic plasticity are not fully understood, particularly in dendritic regions which are beyond the reach of electrode measurements. We combined voltage-sensitive-dye recordings and Ca(2+) imaging of hippocampal CA1 pyramidal neurons to study large regions of the dendritic arbor, including branches of small diameter (distal apical and oblique dendrites). Dendritic membrane potential transients were monitored at high spatial resolution and correlated with supra-linear [Ca(2+)](i) changes during one cycle of a repetitive patterned stimulation protocol that typically results in the induction of long-term potentiation (LTP). While the increase in the peak membrane depolarization during coincident pre- and post-synaptic activity was required for the induction of supra-linear [Ca(2+)](i) signals shown to be necessary for LTP, the change in the baseline-to-peak amplitude of the backpropagating dendritic action potential (bAP) was not critical in this process. At different dendritic locations, the baseline-to-peak amplitude of the bAP could be either increased, decreased or unaltered at sites where EPSP-AP pairing evoked supra-linear summation of [Ca(2+)](i) transients. We suggest that modulations in the bAP baseline-to-peak amplitude by local EPSPs act as a mechanism that brings the membrane potential into the optimal range for Ca(2+) influx through NMDA receptors (0 to -15 mV); this may require either boosting or the reduction of the bAP, depending on the initial size of both signals.

Imaging of spiking and subthreshold activity of mitral cells with voltage-sensitive dyes.

To obtain a more complete description of individual neurons, it is necessary to complement electrical measurements with technologies such as voltage imaging with intracellular dyes, which permit massive parallel recording from many sites on neuronal processes. Utilizing such an approach, we investigate the functional structure of the mitral cell, the principal output neuron in the rat olfactory bulb. These experiments were designed to determine the number, location, and the stability of spike trigger zones, the excitability of terminal dendritic branches, the pattern and nature of spike initiation and propagation in the primary dendrite, the basic characteristics of the evoked EPSPs at the site of origin (the glomerular tuft), and its attenuation along the primary dendrite. The images of spike trigger zones showed that an action potential can be initiated in three different compartments of the mitral cell: the soma-axon region, the primary dendrite trunk, and the terminal dendritic tuft, which appears to be fully excitable. The amplitude of the EPSPs evoked by olfactory nerve stimulation was determined at the site of origin (glomerular tuft) and its attenuation was monitored optically along the entire length of the primary dendrite.

Determinants of low EPSP attenuation in primary dendrites of mitral cells: modeling study.

Efficiency of synaptic potential propagation through neurons depends mainly on their membrane properties and intracellular resistivity. We use a morphologically realistic compartmental model of a mitral cell and data obtained from whole-cell patch-clamp and voltage-imaging experiments to explore passive parameter space in which reported low EPSP attenuation is observed.

Imaging brain activity with voltage- and calcium-sensitive dyes.

This paper presents three examples of imaging brain activity with voltage- or calcium-sensitive dyes and then discusses the methodological aspects of the measurements that are needed to achieve an optimal signal-to-noise ratio. Internally injected voltage-sensitive dye can be used to monitor membrane potential in the dendrites of invertebrate and vertebrate neurons in in vitro preparations. Both invertebrate and vertebrate ganglia can be bathed in voltage-sensitive dyes to stain all of the cell bodies in the preparation. These dyes can then be used to follow the spike activity of many neurons simultaneously while the preparations are generating behaviors. Calcium-sensitive dyes that are internalized into olfactory receptor neurons in the nose will, after several days, be transported to the nerve terminals of these cells in the olfactory bulb. There they can be used to measure the input from the nose to the bulb. Three kinds of noise are discussed. a. Shot noise from the random emission of photons from the preparation. b. Vibrational noise from external sources. c. Noise that occurs in the absence of light, the dark noise. Three different parts of the light measuring apparatus are discussed: the light sources, the optics, and the cameras. The major effort presently underway to improve the usefulness of optical recordings of brain activity are to find methods for staining individual cell types in the brain. Most of these efforts center around fluorescent protein sensors of activity.

Voltage imaging from dendrites of mitral cells: EPSP attenuation and spike trigger zones.

To obtain a more complete description of individual neurons, it is necessary to complement the electrical patch pipette measurements with technologies that permit a massive parallel recording from many sites on neuronal processes. This can be achieved by using voltage imaging with intracellular dyes. With this approach, we investigated the functional structure of a mitral cell, the principal output neuron in the rat olfactory bulb. The most significant finding concerns the characteristics of EPSPs at the synaptic sites and surprisingly small attenuation along the trunk of the primary dendrite. Also, the experiments were performed to determine the number, location, and stability of spike trigger zones, the excitability of terminal dendritic branches, and the pattern and nature of spike initiation and propagation in the primary and secondary dendrites. The results show that optical data can be used to deduce the amplitude and shape of the EPSPs evoked by olfactory nerve stimulation at the site of origin (glomerular tuft) and to determine its attenuation along the entire length of the primary dendrite. This attenuation corresponds to an unusually large mean apparent "length constant" of the primary dendrite. Furthermore, the images of spike trigger zones showed that an action potential can be initiated in three different compartments of the mitral cell: the soma-axon region, the primary dendrite trunk, and the terminal dendritic tuft, which appears to be fully excitable. Finally, secondary dendrites clearly support the active propagation of action potentials.