Long-term potentiation expands information content of hippocampal dentate gyrus synapses.

An approach combining signal detection theory and precise 3D reconstructions from serial section electron microscopy (3DEM) was used to investigate synaptic plasticity and information storage capacity at medial perforant path synapses in adult hippocampal dentate gyrus in vivo. Induction of long-term potentiation (LTP) markedly increased the frequencies of both small and large spines measured 30 minutes later. This bidirectional expansion resulted in heterosynaptic counterbalancing of total synaptic area per unit length of granule cell dendrite. Control hemispheres exhibited 6.5 distinct spine sizes for 2.7 bits of storage capacity while LTP resulted in 12.9 distinct spine sizes (3.7 bits). In contrast, control hippocampal CA1 synapses exhibited 4.7 bits with much greater synaptic precision than either control or potentiated dentate gyrus synapses. Thus, synaptic plasticity altered total capacity, yet hippocampal subregions differed dramatically in their synaptic information storage capacity, reflecting their diverse functions and activation histories.

Synapse-specific structural plasticity that protects and refines local circuits during LTP and LTD.

Synapses form trillions of connections in the brain. Long-term potentiation (LTP) and long-term depression (LTD) are cellular mechanisms vital for learning that modify the strength and structure of synapses. Three-dimensional reconstruction from serial section electron microscopy reveals three distinct pre- to post-synaptic arrangements: strong active zones (AZs) with tightly docked vesicles, weak AZs with loose or non-docked vesicles, and nascent zones (NZs) with a postsynaptic density but no presynaptic vesicles. Importantly, LTP can be temporarily saturated preventing further increases in synaptic strength. At the onset of LTP, vesicles are recruited to NZs, converting them to AZs. During recovery of LTP from saturation (1-4 h), new NZs form, especially on spines where AZs are most enlarged by LTP. Sentinel spines contain smooth endoplasmic reticulum (SER), have the largest synapses and form clusters with smaller spines lacking SER after LTP recovers. We propose a model whereby NZ plasticity provides synapse-specific AZ expansion during LTP and loss of weak AZs that drive synapse shrinkage during LTD. Spine clusters become functionally engaged during LTP or disassembled during LTD. Saturation of LTP or LTD probably acts to protect recently formed memories from ongoing plasticity and may account for the advantage of spaced over massed learning. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Long-Term Potentiation Produces a Sustained Expansion of Synaptic Information Storage Capacity in Adult Rat Hippocampus.

Long-term potentiation (LTP) has become a standard model for investigating synaptic mechanisms of learning and memory. Increasingly, it is of interest to understand how LTP affects the synaptic information storage capacity of the targeted population of synapses. Here, structural synaptic plasticity during LTP was explored using three-dimensional reconstruction from serial section electron microscopy. Storage capacity was assessed by applying a new analytical approach, Shannon information theory, to delineate the number of functionally distinguishable synaptic strengths. LTP was induced by delta-burst stimulation of perforant pathway inputs to the middle molecular layer of hippocampal dentate granule cells in adult rats. Spine head volumes were measured as predictors of synaptic strength and compared between LTP and control hemispheres at 30 min and 2 hr after the induction of LTP. Synapses from the same axon onto the same dendrite were used to determine the precision of synaptic plasticity based on the similarity of their physical dimensions. Shannon entropy was measured by exploiting the frequency of spine heads in functionally distinguishable sizes to assess the degree to which LTP altered the number of bits of information storage. Outcomes from these analyses reveal that LTP expanded storage capacity; the distribution of spine head volumes was increased from 2 bits in controls to 3 bits at 30 min and 2.7 bits at 2 hr after the induction of LTP. Furthermore, the distribution of spine head volumes was more uniform across the increased number of functionally distinguishable sizes following LTP, thus achieving more efficient use of coding space across the population of synapses.

Violation of the ultrastructural size principle in the dorsolateral prefrontal cortex underlies working memory impairment in the aged common marmoset (Callithrix jacchus).

Morphology and function of the dorsolateral prefrontal cortex (dlPFC), and corresponding working memory performance, are affected early in the aging process, but nearly half of aged individuals are spared of working memory deficits. Translationally relevant model systems are critical for determining the neurobiological drivers of this variability. The common marmoset (Callithrix jacchus) is advantageous as a model for these investigations because, as a non-human primate, marmosets have a clearly defined dlPFC that enables measurement of prefrontal-dependent cognitive functions, and their short (∼10 year) lifespan facilitates longitudinal studies of aging. Previously, we characterized working memory capacity in a cohort of marmosets that collectively covered the lifespan, and found age-related working memory impairment. We also found a remarkable degree of heterogeneity in performance, similar to that found in humans. Here, we tested the hypothesis that changes to synaptic ultrastructure that affect synaptic efficacy stratify marmosets that age with cognitive impairment from those that age without cognitive impairment. We utilized electron microscopy to visualize synapses in the marmoset dlPFC and measured the sizes of boutons, presynaptic mitochondria, and synapses. We found that coordinated scaling of the sizes of synapses and mitochondria with their associated boutons is essential for intact working memory performance in aged marmosets. Further, lack of synaptic scaling, due to a remarkable failure of synaptic mitochondria to scale with presynaptic boutons, selectively underlies age-related working memory impairment. We posit that this decoupling results in mismatched energy supply and demand, leading to impaired synaptic transmission. We also found that aged marmosets have fewer synapses in dlPFC than young, though the severity of synapse loss did not predict whether aging occurred with or without cognitive impairment. This work identifies a novel mechanism of synapse dysfunction that stratifies marmosets that age with cognitive impairment from those that age without cognitive impairment. The process by which synaptic scaling is regulated is yet unknown and warrants future investigation.

Dually innervated dendritic spines develop in the absence of excitatory activity and resist plasticity through tonic inhibitory crosstalk.

Dendritic spines can be directly connected to both inhibitory and excitatory presynaptic terminals, resulting in nanometer-scale proximity of opposing synaptic functions. While dually innervated spines (DiSs) are observed throughout the central nervous system, their developmental timeline and functional properties remain uncharacterized. Here we used a combination of serial section electron microscopy, live imaging, and local synapse activity manipulations to investigate DiS development and function in rodent hippocampus. Dual innervation occurred early in development, even on spines where the excitatory input was locally silenced. Synaptic NMDA receptor currents were selectively reduced at DiSs through tonic GABAB receptor signaling. Accordingly, spine enlargement normally associated with long-term potentiation on singly innervated spines (SiSs) was blocked at DiSs. Silencing somatostatin interneurons or pharmacologically blocking GABABRs restored NMDA receptor function and structural plasticity to levels comparable to neighboring SiSs. Thus, hippocampal DiSs are stable structures where function and plasticity are potently regulated by nanometer-scale GABAergic signaling.

Dendritic Spine Density Scales with Microtubule Number in Rat Hippocampal Dendrites.

Microtubules deliver essential resources to and from synapses. Three-dimensional reconstructions in rat hippocampus reveal a sampling bias regarding spine density that needs to be controlled for dendrite caliber and resource delivery based on microtubule number. The strength of this relationship varies across dendritic arbors, as illustrated for area CA1 and dentate gyrus. In both regions, proximal dendrites had more microtubules than distal dendrites. For CA1 pyramidal cells, spine density was greater on thicker than thinner dendrites in stratum radiatum, or on the more uniformly thin terminal dendrites in stratum lacunosum moleculare. In contrast, spine density was constant across the cone shaped arbor of tapering dendrites from dentate granule cells. These differences suggest that thicker dendrites supply microtubules to subsequent dendritic branches and local dendritic spines, whereas microtubules in thinner dendrites need only provide resources to local spines. Most microtubules ran parallel to dendrite length and associated with long, presumably stable mitochondria, which occasionally branched into lateral dendritic branches. Short, presumably mobile, mitochondria were tethered to microtubules that bent and appeared to direct them into a thin lateral branch. Prior work showed that dendritic segments with the same number of microtubules had elevated resources in subregions of their dendritic shafts where spine synapses had enlarged, and spine clusters had formed. Thus, additional microtubules were not required for redistribution of resources locally to growing spines or synapses. These results provide new understanding about the potential for microtubules to regulate resource delivery to and from dendritic branches and locally among dendritic spines.

Ultrastructure of light-activated axons following optogenetic stimulation to produce late-phase long-term potentiation.

Analysis of neuronal compartments has revealed many state-dependent changes in geometry but establishing synapse-specific mechanisms at the nanoscale has proven elusive. We co-expressed channelrhodopsin2-GFP and mAPEX2 in a subset of hippocampal CA3 neurons and used trains of light to induce late-phase long-term potentiation (L-LTP) in area CA1. L-LTP was shown to be specific to the labeled axons by severing CA3 inputs, which prevented back-propagating recruitment of unlabeled axons. Membrane-associated mAPEX2 tolerated microwave-enhanced chemical fixation and drove tyramide signal amplification to deposit Alexa Fluor dyes in the light-activated axons. Subsequent post-embedding immunogold labeling resulted in outstanding ultrastructure and clear distinctions between labeled (activated), and unlabeled axons without obscuring subcellular organelles. The gold-labeled axons in potentiated slices were reconstructed through serial section electron microscopy; presynaptic vesicles and other constituents could be quantified unambiguously. The genetic specification, reliable physiology, and compatibility with established methods for ultrastructural preservation make this an ideal approach to link synapse ultrastructure and function in intact circuits.

Structural plasticity of dendritic secretory compartments during LTP-induced synaptogenesis.

Long-term potentiation (LTP), an increase in synaptic efficacy following high-frequency stimulation, is widely considered a mechanism of learning. LTP involves local remodeling of dendritic spines and synapses. Smooth endoplasmic reticulum (SER) and endosomal compartments could provide local stores of membrane and proteins, bypassing the distant Golgi apparatus. To test this hypothesis, effects of LTP were compared to control stimulation in rat hippocampal area CA1 at postnatal day 15 (P15). By two hours, small spines lacking SER increased after LTP, whereas large spines did not change in frequency, size, or SER content. Total SER volume decreased after LTP consistent with transfer of membrane to the added spines. Shaft SER remained more abundant in spiny than aspiny dendritic regions, apparently supporting the added spines. Recycling endosomes were elevated specifically in small spines after LTP. These findings suggest local secretory trafficking contributes to LTP-induced synaptogenesis and primes the new spines for future plasticity.

Localization and expression of group I metabotropic glutamate receptors in the mouse striatum, globus pallidus, and subthalamic nucleus: regulatory effects of MPTP treatment and constitutive Homer deletion.

Group I metabotropic glutamate receptors (mGluRs), mGluR1 and mGluR5, regulate activity in the globus pallidus (GP) and subthalamic nucleus (STN). To test whether the localization of group I mGluRs is altered in parkinsonism, we used immunoelectron microscopy to analyze the subcellular and subsynaptic distribution of mGluR1a and mGluR5 in GP and STN of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. Homer1 and Homer2 knock-out mice were used to assess the role of Homer in MPTP-induced redistribution of group I mGluRs. We also examined the effects of MPTP on the expression levels of group I mGluRs and Homer proteins in GP and striatum. MPTP treatment significantly reduced the expression levels of H1a and mGluR1a in striatum but not in GP. Although light microscopy did not reveal noticeable effects of MPTP treatment on the distribution of group I mGluRs and Homer proteins in GP and STN, specific changes in the ultrastructural localization of mGluR1a were found in MPTP-treated normal and Homer knock-out mice. An increase in the expression of presynaptic axonal and terminal mGluR1a labeling and an increased level of mGluR1a immunoreactivity in the postsynaptic specialization of putative GABAergic synapses were among the most significant effects induced by dopamine depletion. However, neither of these changes was found for mGluR5, which, in contrast, displayed complex regulatory alterations in its subsynaptic distribution in response to Homer deletion and MPTP lesion. Thus, nigrostriatal dopaminergic lesion and Homer deletion lead to changes in the trafficking of group I mGluRs in vivo that are specific to receptor subtypes and brain areas.

Astrocytic and neuronal localization of the scaffold protein Na+/H+ exchanger regulatory factor 2 (NHERF-2) in mouse brain.

The Na+/H+ exchanger regulatory factor 2 (NHERF-2) is a scaffold protein that regulates cellular signaling by forming protein complexes. Several proteins known to interact with NHERF-2 are abundantly expressed in the central nervous system, but little is known about NHERF-2 localization in the brain. By using immunohistochemistry combined with light and electron microscopy, we found that many populations of astrocytes, as well as some populations of neurons, were immunopositive for NHERF-2 throughout the mouse brain. Quantitative analysis of the subcellular distribution of NHERF-2 immunostaining in four brain structures, cerebral cortex, hippocampus, striatum, and cerebellar cortex, showed that NHERF-2 was expressed mainly in astrocytic processes but was also sometimes observed in both pre- and postsynaptic neuronal elements. NHERF-2 immunostaining was associated mainly with the plasma membrane of neurons and astrocytes. However, NHERF-2 immunoreactivity was also observed in association with synaptic vesicles in putative glutamatergic axon terminals. The subcellular localization of NHERF-2 in brain is consistent with a role for NHERF-2 in forming complexes between cell surface and cytosolic proteins, and the preferential expression of NHERF-2 in astrocytes suggests that this scaffold protein may play an important role in astrocytic physiology.

An electron microscope immunocytochemical study of GABA(B) R2 receptors in the monkey basal ganglia: a comparative analysis with GABA(B) R1 receptor distribution.

Functional gamma-aminobutyric acid (GABA)(B) receptors are heterodimers made up of GABA(B) R1 and GABA(B) R2 subunits. The subcellular localization of GABA(B) R2 receptors remains poorly known in the central nervous system. Therefore, we performed an ultrastructural analysis of the localization of GABA(B) R2 receptor immunoreactivity in the monkey basal ganglia. Furthermore, to characterize better the neuronal sites at which GABA(B) R1 and GABA(B) R2 may interact to form functional receptors, we compared the relative distribution of immunoreactivity of the two GABA(B) receptors in various basal ganglia nuclei. Light to moderate GABA(B) R2 immunoreactivity was found in cell bodies and neuropil elements in all basal ganglia nuclei. At the electron microscope level, GABA(B) R2 immunoreactivity was commonly expressed postsynaptically, although immunoreactive preterminal axonal segments were also frequently encountered, particularly in the globus pallidus and substantia nigra, where they accounted for the third of the total number of GABA(B) R2-containing elements. A few labeled terminals that displayed the ultrastructural features of glutamatergic boutons were occasionally found in most basal ganglia nuclei, except for the subthalamic nucleus, which was devoid of GABA(B) R2-immunoreactive boutons. The relative distribution of GABA(B) R2 immunoreactivity in the monkey basal ganglia was largely consistent with that of GABA(B) R1, but some exceptions were found, most noticeably in the globus pallidus and substantia nigra, which contained a significantly larger proportion of presynaptic elements labeled for GABA(B) R1 than GABA(B) R2. These findings suggest the possible coexistence and heterodimerization of GABA(B) R1 and GABA(B) R2 at various pre- and postsynaptic sites, but also raise the possibility that the formation of functional GABA(B) receptors in specific compartments of basal ganglia neurons relies on mechanisms other than GABA(B) R1/R2 heterodimerization.

Subcellular and subsynaptic localization of group I metabotropic glutamate receptors in the monkey subthalamic nucleus.

Both subtypes of group I metabotropic glutamate receptor, mGluR1 and mGluR5, are expressed postsynaptically in neurons of the subthalamic nucleus (STN), and their activation induces different physiological responses. To test whether these effects could be explained by a differential localization of the two group I mGluRs, we analyzed the subcellular and subsynaptic distribution of mGluR1a and mGluR5 in the monkey STN. Double-immunofluorescence and light microscopic analyses revealed that both group I mGluR subtypes were strongly coexpressed in the neuropil and neuronal perikarya. Astrocytic perikarya exhibited intense mGluR1a, but no detectable mGluR5, immunoreactivity. At the electron microscopic level, immunoperoxidase labeling for both mGluR1a and mGluR5 was localized mainly in dendrites. A significant proportion of the total pool of mGluR1a-immunoreactive elements was accounted for by glial cell processes, whereas glial cell labeling was much less frequently encountered in sections immunostained for mGluR5. Preembedding immunogold labeling in STN dendrites revealed that 60-70% of the gold labeling for both mGluR subtypes was intracellular, whereas 30-40% was apposed to the plasma membrane. Of the plasma membrane-apposed particles, more than 90% were extrasynaptic; fewer than 10% were associated with symmetric or asymmetric synapses. Most of the synapse-associated labeling was found at the edges of both asymmetric and symmetric postsynaptic specializations. Some extrasynaptic gold particles were aggregated on parts of the plasma membrane tightly apposed by glial processes. These findings demonstrate that mGluR1a and mGluR5 exhibit a similar pattern of subsynaptic localization in monkey STN neurons, with both receptor subtypes exhibiting substantial extrasynaptic and perisynaptic localization.

Amphetamine-induced behavioral activation is associated with variable changes in basal ganglia output neurons recorded from awake, behaving rats.

Systemic or intra-striatal administration of d-amphetamine (AMPH) elicits a dose-dependent pattern of behavioral activation and neuronal firing in the striatum. To determine if the AMPH-induced striatal firing pattern is expressed in the substantia nigra pars reticulata (SNr), a main target of striatal efferents and the primary output nucleus of the basal ganglia, we recorded the activity of 214 SNr units in alert, behaving rats responding to either systemic (1.0 or 5.0 mg/kg, sc) or intra-striatal (20 microg/microl/min) AMPH. Both routes of administration increased behavior but the strongest effects occurred after systemic injection. A dose of 1.0 mg/kg progressively increased locomotion, head movements, and sniffing, whereas after 5.0 mg/kg behavioral responding became progressively more focused and stereotyped. The collective response of SNr neurons was a net increase in firing rate that was most apparent after the low systemic dose and intra-striatal infusion. Further analysis revealed significant unit populations that were either excited, inhibited or showed no change. Although excitations predominated over inhibitions in all cases, a sizable population of units was unresponsive: approximately 25% to systemic AMPH and almost half to intra-striatal infusion. Subsequent injection of haloperidol (0.5 or 1.0 mg/kg, sc), a dopamine receptor antagonist, reversed both the behavioral and electrophysiological effects of AMPH. Thus, as in striatum, dopamine appears to play a critical role in AMPH-induced changes in SNr activity. Interestingly, however, SNr activity did not closely parallel the striatal response, suggesting that patterns of neuronal responding to AMPH in striatum are not reliably relayed to SNr.

Dynamics of nascent and active zone ultrastructure as synapses enlarge during long-term potentiation in mature hippocampus.

Nascent zones and active zones are adjacent synaptic regions that share a postsynaptic density, but nascent zones lack the presynaptic vesicles found at active zones. Here dendritic spine synapses were reconstructed through serial section electron microscopy (3DEM) and EM tomography to investigate nascent zone dynamics during long-term potentiation (LTP) in mature rat hippocampus. LTP was induced with theta-burst stimulation, and comparisons were made with control stimulation in the same hippocampal slices at 5 minutes, 30 minutes, and 2 hours post-induction and to perfusion-fixed hippocampus in vivo. Nascent zones were present at the edges of ∼35% of synapses in perfusion-fixed hippocampus and as many as ∼50% of synapses in some hippocampal slice conditions. By 5 minutes, small dense-core vesicles known to transport active zone proteins moved into more presynaptic boutons. By 30 minutes, nascent zone area decreased, without significant change in synapse area, suggesting that presynaptic vesicles were recruited to preexisting nascent zones. By 2 hours, both nascent and active zones were enlarged. Immunogold labeling revealed glutamate receptors in nascent zones; however, average distances from nascent zones to docked presynaptic vesicles ranged from 170 ± 5 nm in perfusion-fixed hippocampus to 251 ± 4 nm at enlarged synapses by 2 hours during LTP. Prior stochastic modeling suggests that decrease in glutamate concentration reduces the probability of glutamate receptor activation from 0.4 at the center of release to 0.1 just 200 nm away. Thus, conversion of nascent zones to functional active zones likely requires the recruitment of presynaptic vesicles during LTP.

Large-volume reconstruction of brain tissue from high-resolution serial section images acquired by SEM-based scanning transmission electron microscopy.

With recent improvements in instrumentation and computational tools, serial section electron microscopy has become increasingly straightforward. A new method for imaging ultrathin serial sections is developed based on a field emission scanning electron microscope fitted with a transmitted electron detector. This method is capable of automatically acquiring high-resolution serial images with a large field size and very little optical and physical distortions. In this chapter, we describe the procedures leading to the generation and analyses of a large-volume stack of high-resolution images (64 µm × 64 µm × 10 µm, or larger, at 2 nm pixel size), including how to obtain large-area serial sections of uniform thickness from well-preserved brain tissue that is rapidly perfusion-fixed with mixed aldehydes, processed with a microwave-enhanced method, and embedded into epoxy resin.

Automated transmission-mode scanning electron microscopy (tSEM) for large volume analysis at nanoscale resolution.

Transmission-mode scanning electron microscopy (tSEM) on a field emission SEM platform was developed for efficient and cost-effective imaging of circuit-scale volumes from brain at nanoscale resolution. Image area was maximized while optimizing the resolution and dynamic range necessary for discriminating key subcellular structures, such as small axonal, dendritic and glial processes, synapses, smooth endoplasmic reticulum, vesicles, microtubules, polyribosomes, and endosomes which are critical for neuronal function. Individual image fields from the tSEM system were up to 4,295 µm(2) (65.54 µm per side) at 2 nm pixel size, contrasting with image fields from a modern transmission electron microscope (TEM) system, which were only 66.59 µm(2) (8.160 µm per side) at the same pixel size. The tSEM produced outstanding images and had reduced distortion and drift relative to TEM. Automated stage and scan control in tSEM easily provided unattended serial section imaging and montaging. Lens and scan properties on both TEM and SEM platforms revealed no significant nonlinear distortions within a central field of ∼100 µm(2) and produced near-perfect image registration across serial sections using the computational elastic alignment tool in Fiji/TrakEM2 software, and reliable geometric measurements from RECONSTRUCT™ or Fiji/TrakEM2 software. Axial resolution limits the analysis of small structures contained within a section (∼45 nm). Since this new tSEM is non-destructive, objects within a section can be explored at finer axial resolution in TEM tomography with current methods. Future development of tSEM tomography promises thinner axial resolution producing nearly isotropic voxels and should provide within-section analyses of structures without changing platforms. Brain was the test system given our interest in synaptic connectivity and plasticity; however, the new tSEM system is readily applicable to other biological systems.