Synaptic Clustering Research Articles

LRRTM2 controls presynapse nano-organization and AMPA receptor sub-positioning through Neurexin-binding interface

Synapses are organized into nanocolumns that control synaptic transmission efficacy through precise alignment of postsynaptic neurotransmitter receptors and presynaptic release sites. Recent evidence show that Leucine-Rich Repeat Transmembrane protein LRRTM2, highly enriched and confined at synapses, interacts with Neurexins through its C-terminal cap, but the role of this binding interface has not been explored in synapse formation and function. Here, we develop a conditional knock-out mouse model (cKO) to address the molecular mechanisms of LRRTM2 regulation, and its role in synapse organization and function. We show that LRRTM2 cKO specifically impairs excitatory synapse formation and function in mice. Surface expression, synaptic clustering, and membrane dynamics of LRRTM2 are tightly controlled by selective motifs in the C-terminal domain. Conversely, the N-terminal domain controls presynapse nano-organization and postsynapse AMPAR sub-positioning and stabilization through the recently identified Neurexin-binding interface. Thus, we identify LRRTM2 as a central organizer of pre- and post- excitatory synapse nanostructure through interaction with presynaptic Neurexins.

Clustering of synaptic engram: Functional and structural basis of memory

Studies on memory engram have demonstrated how experience and learning can be allocated at a neuronal level for centuries. Recently emerging evidence narrowed down further to the synaptic connections and their patterned allocation on dendrites. Notably, groups of synapses within a specific range within dendrites known as ’synaptic clusters’ have been revealed in association with learning and memory. Previous investigations have shown that a variety of factors mediated by both presynaptic inputs and postsynaptic dendrites contribute to clustering. Here, we review the neural mechanism of synaptic clustering and its correlation with memory. We highlight the recent findings about the clustering of synaptic engrams and memory formation and discuss future directions.

Unveiling mitochondria as central components driving cognitive decline in alzheimer's disease through cross-transcriptomic analysis of hippocampus and entorhinal cortex microarray datasets

Alzheimer's disease (AD) is a prevalent neurodegenerative disorder characterized by symptoms such as memory loss and impaired learning. This study conducted a cross-transcriptomic analysis of AD using existing microarray datasets from the hippocampus (HC) and entorhinal cortex (EC), comparing them with age-matched non-AD controls. Both of these brain regions are critical for learning and memory processing and are vulnerable areas that exhibit abnormalities in early AD. The cross-transcriptomic analysis identified 564 significantly differentially expressed genes in HC and 479 in EC. Among these, 151 genes were significantly differentially expressed in both tissues, with functions related to synaptic vesicle clustering, synaptic vesicle exocytosis/endocytosis, mitochondrial ATP synthesis, hydrogen ion transmembrane transport, and structural constituent of cytoskeleton, suggesting a potential association between cognitive decline in AD, synaptic vesicle dynamics, dysregulation of cytoskeleton organization, and mitochondrial dysfunction. Further gene ontology analysis specific to the HC revealed the gene ontology enrichment in aerobic respiration, synaptic vesicle cycle, and oxidative phosphorylation. The enrichment analysis in CA1 of HC revealed differentiation in gene expression related to mitochondrial membrane functions involved in bioenergetics, mitochondrial electron transport, and biological processes associated with microtubule-based process, while analysis in the EC region showed enrichment in synaptic vesicle dynamics which is associated with neurotransmitter release and the regulation of postsynaptic membrane potential and synaptic transmission of GABAergic and glutamatergic synapse. Protein-protein interaction analysis highlighted central hub proteins predominantly expressed in mitochondria, involved in regulation of oxidative stress and ATP synthesis. These hub proteins interact not only within the mitochondria but also with proteins in the vesicular membrane and neuronal cytoskeleton, indicating a central role of mitochondria. This finding underscores the association between clinical symptoms and mitochondrial dysregulation of synaptic vesicle dynamics, cytoskeleton organization, and mitochondrial processes in both the HC and EC of AD. Therefore, targeting these dysregulated pathways could provide promising therapeutic targets aimed at cognitive decline and memory impairment in early AD stages.

Alternative translation initiation produces synaptic organizer proteoforms with distinct localization and functions

While many mRNAs contain more than one translation initiation site (TIS), the functions of most alternative TISs and their corresponding protein isoforms (proteoforms) remain undetermined. Here, we showed that alternative usage of CUG and AUG TISs in neuronal pentraxin receptor (NPR) mRNA produced two proteoforms, of which the ratio was regulated by RNA secondary structure and neuronal activity. Downstream AUG initiation truncated the N-terminal transmembrane domain and produced a secreted NPR proteoform sufficient in promoting synaptic clustering of AMPA-type glutamate receptors. Mutations that altered the ratio of NPR proteoforms reduced AMPA receptors in parvalbumin-positive interneurons and affected learning behaviors in mice. In addition to NPR, upstream AUU-initiated N-terminal extension of C1q-like synaptic organizers anchored these otherwise secreted factors to the membrane. Together, these results uncovered the plasticity of N-terminal signal sequences regulated by alternative TIS usage as a potentially widespread mechanism in diversifying protein localization and functions.

MiRNA-mediated control of gephyrin synthesis drives sustained inhibitory synaptic plasticity.

Activity-dependent protein synthesis is crucial for long-lasting forms of synaptic plasticity. However, our understanding of translational mechanisms controlling GABAergic synapses is limited. One distinct form of inhibitory long-term potentiation (iLTP) enhances postsynaptic clusters of GABAARs and the primary inhibitory scaffold, gephyrin, to promote sustained synaptic strengthening. While we previously found that persistent iLTP requires mRNA translation, the mechanisms controlling plasticity-induced gephyrin translation remain unknown. We identify miR153 as a novel regulator of Gphn mRNA translation which controls gephyrin protein levels and synaptic clustering, ultimately impacting inhibitory synaptic structure and function. iLTP induction downregulates miR153, reversing its translational suppression of Gphn mRNA and promoting de novo gephyrin protein synthesis and synaptic clustering during iLTP. Finally, we find that reduced miR153 expression during iLTP is driven by an excitation-transcription coupling pathway involving calcineurin, NFAT and HDACs, which also controls the miRNA-dependent upregulation of GABAARs. Together, we delineate a miRNA-dependent post-transcriptional mechanism that controls the expression of the key synaptic scaffold, gephyrin, and may converge with parallel miRNA pathways to coordinate gene upregulation to maintain inhibitory synaptic plasticity.

High-Speed Atomic Force Microscopy Reveals Fluctuations and Dimer Splitting of the N-Terminal Domain of GluA2 Ionotropic Glutamate Receptor-Auxiliary Subunit Complex.

α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid glutamate receptors (AMPARs) enable rapid excitatory synaptic transmission by localizing to the postsynaptic density of glutamatergic spines. AMPARs possess large extracellular N-terminal domains (NTDs), which are crucial for AMPAR clustering at synaptic sites. However, the dynamics of NTDs and the molecular mechanism governing their synaptic clustering remain elusive. Here, we employed high-speed atomic force microscopy (HS-AFM) to directly visualize the conformational dynamics of NTDs in the GluA2 subunit complexed with TARP γ2 in lipid environments. HS-AFM videos of GluA2-γ2 in the resting and activated/open states revealed fluctuations in NTD dimers. Conversely, in the desensitized/closed state, the two NTD dimers adopted a separated conformation with less fluctuation. Notably, we observed individual NTD dimers transitioning into monomers, with extended monomeric states in the activated/open state. Molecular dynamics simulations provided further support, confirming the energetic stability of the monomeric NTD states within lipids. This NTD-dimer splitting resulted in subunit exchange between the receptors and increased the number of interaction sites with synaptic protein neuronal pentraxin 1 (NP1). Moreover, our HS-AFM studies revealed that NP1 forms a ring-shaped octamer through N-terminal disulfide bonds and binds to the tip of the NTD. These findings suggest a molecular mechanism in which NP1, upon forming an octamer, is secreted into the synaptic region and binds to the tip of the GluA2 NTD, thereby bridging and clustering multiple AMPARs. Thus, our findings illuminate the critical role of NTD dynamics in the synaptic clustering of AMPARs and contribute valuable insights into the fundamental processes of synaptic transmission.

N-acetylation of α-synuclein enhances synaptic vesicle clustering mediated by α-synuclein and lysophosphatidylcholine.

Post-translational modifications (PTMs) of α-synuclein (α-syn) such as acetylation and phosphorylation play important yet distinct roles in regulating α-syn conformation, membrane binding, and amyloid aggregation. However, how PTMs regulate α-syn function in presynaptic terminals remains unclear. Previously, we reported that α-syn clusters synaptic vesicles (SV)1, and neutral phospholipid lysophosphatidylcholine (LPC) can mediate this clustering2. Here, based on our previous findings, we further demonstrate that N-terminal acetylation, which occurs under physiological conditions and is irreversible in mammalian cells, significantly enhances the functional activity of α-syn in clustering SVs. Mechanistic studies reveal that this enhancement is caused by the N-acetylation-promoted insertion of α-syn's N-terminus and increased intermolecular interactions on the LPC-containing membrane. Our work demonstrates that N-acetylation fine-tunes α-syn-LPC interaction for mediating α-syn's function in SV clustering.

Distinct active zone protein machineries mediate Ca2+ channel clustering and vesicle priming at hippocampal synapses.

Action potentials trigger neurotransmitter release at the presynaptic active zone with spatiotemporal precision. This is supported by protein machinery that mediates synaptic vesicle priming and clustering of CaV2 Ca2+ channels nearby. One model posits that scaffolding proteins directly tether vesicles to CaV2s; however, here we find that at mouse hippocampal synapses, CaV2 clustering and vesicle priming are executed by separate machineries. CaV2 nanoclusters are positioned at variable distances from those of the priming protein Munc13. The active zone organizer RIM anchors both proteins but distinct interaction motifs independently execute these functions. In transfected cells, Liprin-α and RIM form co-assemblies that are separate from CaV2-organizing complexes. At synapses, Liprin-α1-Liprin-α4 knockout impairs vesicle priming but not CaV2 clustering. The cell adhesion protein PTPσ recruits Liprin-α, RIM and Munc13 into priming complexes without co-clustering CaV2s. We conclude that active zones consist of distinct machineries to organize CaV2s and prime vesicles, and Liprin-α and PTPσ specifically support priming site assembly.

Functional and Structural Changes in Diaphragm Neuromuscular Junctions in Early Aging.

Age-related impairment of the diaphragm causes respiratory complications. Neuromuscular junction (NMJ) dysfunction can be one of the triggering events in diaphragm weaknesses in old age. Prominent structural and functional alterations in diaphragm NMJs were described in elderly rodents, but NMJ changes in middle age remain unclear. Here, we compared diaphragm muscles from young adult (3 months) and middle-aged (12 months) BALB/c mice. Microelectrode recordings, immunofluorescent staining, electron microscopy, myography, and whole-body plethysmography were used. We revealed presynaptic (i) and postsynaptic (ii) changes. The former (i) included an increase in both action potential propagation velocity and neurotransmitter release evoked by low-, moderate-, and high-frequency activity but a decrease in immunoexpression of synapsin 1 and synaptic vesicle clustering. The latter (ii) consisted of a decrease in currents via nicotinic acetylcholine receptors and the area of their distribution. These NMJ changes correlated with increased contractile responses to moderate- to high-frequency nerve activation. Additionally, we found alterations in the pattern of respiration (an increase in peak inspiratory flow and a tendency of elevation of the tidal volume), which imply increased diaphragm activity in middle-aged mice. We conclude that enhancement of neuromuscular communication (due to presynaptic mechanism) accompanied by improved contractile responses occurs in the diaphragm in early aging.

γ1 GABAA Receptors in Spinal Nociceptive Circuits.

GABAergic neurons and GABAA receptors (GABAARs) are critical elements of almost all neuronal circuits. Most GABAARs of the CNS are heteropentameric ion channels composed of two α, two β, and one γ subunits. These receptors serve as important drug targets for benzodiazepine (BDZ) site agonists, which potentiate the action of GABA at GABAARs. Most GABAAR classifications rely on the heterogeneity of the α subunit (α1-α6) included in the receptor complex. Heterogeneity of the γ subunits (γ1-γ3), which mediate synaptic clustering of GABAARs and contribute, together with α subunits, to the benzodiazepine (BDZ) binding site, has gained less attention, mainly because γ2 subunits greatly outnumber the other γ subunits in most brain regions. Here, we have investigated a potential role of non-γ2 GABAARs in neural circuits of the spinal dorsal horn, a key site of nociceptive processing. Female and male mice were studied. We demonstrate that besides γ2 subunits, γ1 subunits are significantly expressed in the spinal dorsal horn, especially in its superficial layers. Unlike global γ2 subunit deletion, which is lethal, spinal cord-specific loss of γ2 subunits was well tolerated. GABAAR clustering in the superficial dorsal horn remained largely unaffected and antihyperalgesic actions of HZ-166, a nonsedative BDZ site agonist, were partially retained. Our results thus suggest that the superficial dorsal horn harbors functionally relevant amounts of γ1 subunits that support the synaptic clustering of GABAARs in this site. They further suggest that γ1 containing GABAARs contribute to the spinal control of nociceptive information flow.

Nanoscale organization is changed in native, surface AMPARs by mouse brain region and tauopathy.

Synaptic AMPA receptors (AMPARs) on neuronal plasma membranes are correlated with learning and memory. Using a unique labeling and super-resolution imaging, we have visualized the nanoscale synaptic and extra-synaptic organization of native surface AMPARs for the first time in mouse brain slices as a function of brain region and tauopathy. We find that the fraction of surface AMPARs organized in synaptic clusters is two-times smaller in the hippocampus compared to the motor and somatosensory cortex. In 6 months old PS19 model of tauopathy, synaptic and extrasynaptic distributions are disrupted in the hippocampus but not in the cortex. Thus, this optimized super-resolution imaging tool allows us to observe synaptic deterioration at the onset of tauopathy before apparent neurodegeneration.

Clustered synapses develop in distinct dendritic domains in visual cortex before eye opening.

Synaptic inputs to cortical neurons are highly structured in adult sensory systems, such that neighboring synapses along dendrites are activated by similar stimuli. This organization of synaptic inputs, called synaptic clustering, is required for high-fidelity signal processing, and clustered synapses can already be observed before eye opening. However, how clustered inputs emerge during development is unknown. Here, we employed concurrent in vivo whole-cell patch-clamp and dendritic calcium imaging to map spontaneous synaptic inputs to dendrites of layer 2/3 neurons in the mouse primary visual cortex during the second postnatal week until eye opening. We found that the number of functional synapses and the frequency of transmission events increase several fold during this developmental period. At the beginning of the second postnatal week, synapses assemble specifically in confined dendritic segments, whereas other segments are devoid of synapses. By the end of the second postnatal week, just before eye opening, dendrites are almost entirely covered by domains of co-active synapses. Finally, co-activity with their neighbor synapses correlates with synaptic stabilization and potentiation. Thus, clustered synapses form in distinct functional domains presumably to equip dendrites with computational modules for high-capacity sensory processing when the eyes open.

A role for synapsin tetramerization in synaptic vesicle clustering.

Although synapsins have long been proposed to be key regulators of synaptic vesicle (SV) clustering, their mechanism of action has remained mysterious and somewhat controversial. Here, we review synapsins and their associations with each other and with SVs. We highlight the recent hypothesis that synapsin tetramerization is a mechanism for SV clustering. This hypothesis, which aligns with numerous experimental results, suggests that the larger size of synapsin tetramers, in comparison to dimers, allows tetramers to form optimal bridges between SVs that overcome the repulsive force associated with the negatively charged membrane of SVs and allow synapsins to form a reserve pool of SVs within presynaptic terminals.

Excitatory synaptic structural abnormalities produced by templated aggregation of α-syn in the basolateral amygdala

Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are characterized by neuronal α-synuclein (α-syn) inclusions termed Lewy Pathology, which are abundant in the amygdala. The basolateral amygdala (BLA), in particular, receives projections from the thalamus and cortex. These projections play a role in cognition and emotional processing, behaviors which are impaired in α-synucleinopathies. To understand if and how pathologic α-syn impacts the BLA requires animal models of α-syn aggregation. Injection of α-syn pre-formed fibrils (PFFs) into the striatum induces robust α-syn aggregation in excitatory neurons in the BLA that corresponds with reduced contextual fear conditioning. At early time points after aggregate formation, cortico-amygdala excitatory transmission is abolished. The goal of this project was to determine if α-syn inclusions in the BLA induce synaptic degeneration and/or morphological changes. In this study, we used C57BL/6 J mice injected bilaterally with PFFs in the dorsal striatum to induce α-syn aggregate formation in the BLA. A method was developed using immunofluorescence and three-dimensional reconstruction to analyze excitatory cortico-amygdala and thalamo-amygdala presynaptic terminals closely juxtaposed to postsynaptic densities. The abundance and morphology of synapses were analyzed at 6- or 12-weeks post-injection of PFFs. α-Syn aggregate formation in the BLA did not cause a significant loss of synapses, but cortico-amygdala and thalamo-amygdala presynaptic terminals and postsynaptic densities with aggregates of α-syn show increased volumes, similar to previous findings in human DLB cortex, and in non-human primate models of PD. Transmission electron microscopy showed that asymmetric synapses in mice with PFF-induced α-syn aggregates have reduced synaptic vesicle intervesicular distances, similar to a recent study showing phospho-serine-129 α-syn increases synaptic vesicle clustering. Thus, pathologic α-syn causes major alterations to synaptic architecture in the BLA, potentially contributing to behavioral impairment and amygdala dysfunction observed in synucleinopathies.

The distribution and structural plasticity of excitatory synapses along interneuron dendrites in the adult hippocampus

Hippocampal interneurons form a large, heterogeneous group with differing structural, physiological, and chemical phenotypes. Most CA1 interneuron dendrites, however, lack spines. To investigate structural synaptic plasticity along interneuron dendrites, we generated three-dimensional (3D) reconstructions from serial section electron microscopy (EM) of excitatory synapses along aspiny dendrites in CA1 stratum radiatum of young adult rats under control conditions and at 2 h during LTP induced with theta-burst stimulation. In total, 43 aspiny dendritic segments and their synapses were identified and reconstructed in 3D. Aspiny dendrites were categorized as smooth or varicose based on dendritic volume contours along each segment. We further sub-segmented varicose dendrites varicosities and inter-varicose regions using automated and manual methods. We found synapses and total synaptic input per length were distributed uniformly along smooth dendrites. Synapses along varicose dendrites, however, occurred preferentially at varicosities. While synaptic input per length along varicosities was similar to that along smooth dendrites, both synapse number and total synaptic input per length along inter-varicose regions were lower. Dendritic segments were also analyzed for mitochondria and glycogen content. We found that mitochondria and glycogen were also distributed uniformly along smooth dendrites. In varicose dendrites, however, mitochondrial volume and glycogen granule numbers were highest in varicosities, while inter-varicose regions were comparatively devoid of these resources. The spatial distribution of synapses and dendritic resources in both smooth and varicose dendrites was preserved at 2 h during LTP when compared to control conditions. These findings suggest that synapses occur along interneuron dendrites where dendritic resources are most available. When dendritic resources are distributed uniformly throughout the dendrite, as occurs in smooth dendrites, synapse distribution is also uniform. Varicosities, on the other hand, represent resource-rich sites along varicose dendrites where synaptic clustering occurs. This work was supported by the National Science Foundation (NSF 1707356, NSF 2014862) and the National Institutes of Health (R01 5R01MH09598). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Engram mechanisms of memory linking and identity.

Memories are thought to be stored in neuronal ensembles referred to as engrams. Studies have suggested that when two memories occur in quick succession, a proportion of their engrams overlap and the memories become linked (in a process known as prospective linking) while maintaining their individual identities. In this Review, we summarize the key principles of memory linking through engram overlap, as revealed by experimental and modelling studies. We describe evidence of the involvement of synaptic memory substrates, spine clustering and non-linear neuronal capacities in prospective linking, and suggest a dynamic somato-synaptic model, in which memories are shared between neurons yet remain separable through distinct dendritic and synaptic allocation patterns. We also bring into focus retrospective linking, in which memories become associated after encoding via offline reactivation, and discuss key temporal and mechanistic differences between prospective and retrospective linking, as well as the potential differences in their cognitive outcomes.

The impact of canonical Wnt transcriptional repressors TLE3 and TLE4 on postsynaptic transcription at the neuromuscular junction.

Here, we investigated the role of the canonical Wnt signaling pathway transcriptional regulators at the neuromuscular junction. Upon applying a denervation paradigm, the transcription levels of Ctnnb1, Tcf7l1, Tle1, Tle2, Tle3, and Tle4 were significantly downregulated. A significant decrease in canonical Wnt signaling activity was observed using the denervation paradigm in Axin2-lacZ reporter mice. Alterations in the transcriptional profile of the myogenic lineage in response to agrin (AGRN) suggested that TLE3 and TLE4, family members of groucho transducin-like enhancer of split 3 (TLE3), transcriptional repressors known to antagonize T cell factor/lymphoid enhancer factor (TCF)-mediated target gene activation, could be important regulators of canonical Wnt signaling activity at the postsynapse. Knockouts of these genes using CRISPR/Cas9 gene editing in primary skeletal muscle stem cells, called satellite cells, led to decreased AGRN-dependent acetylcholine receptor (CHRN) clustering and reduced synaptic gene transcription upon differentiation of these cells. Overall, our findings demonstrate that TLE3 and TLE4 participate in diminishing canonical Wnt signaling activity, supporting transcription of synaptic genes and CHRN clustering at the neuromuscular junction.

Local and global predictors of synapse elimination during motor learning.

During learning, synaptic connections between excitatory neurons in the brain display considerable dynamism, with new connections being added and old connections eliminated. Synapse elimination offers an opportunity to understand the features of synapses that the brain deems dispensable. However, with limited observations of synaptic activity and plasticity in vivo, the features of synapses subjected to elimination remain poorly understood. Here, we examined the functional basis of synapse elimination in the apical dendrites of L2/3 neurons in the primary motor cortex throughout motor learning. We found no evidence that synapse elimination is facilitated by a lack of activity or other local forms of plasticity. Instead, eliminated synapses display asynchronous activity with nearby synapses, suggesting that functional synaptic clustering is a critical component of synapse survival. In addition, eliminated synapses show delayed activity timing with respect to postsynaptic output. Thus, synaptic inputs that fail to be co-active with their neighboring synapses or are mistimed with neuronal output are targeted for elimination.

Activity-dependent mitochondrial ROS signaling regulates recruitment of glutamate receptors to synapses.

Our understanding of mitochondrial signaling in the nervous system has been limited by the technical challenge of analyzing mitochondrial function in vivo. In the transparent genetic model Caenorhabditis elegans, we were able to manipulate and measure mitochondrial reactive oxygen species (mitoROS) signaling of individual mitochondria as well as neuronal activity of single neurons in vivo. Using this approach, we provide evidence supporting a novel role for mitoROS signaling in dendrites of excitatory glutamatergic C. elegans interneurons. Specifically, we show that following neuronal activity, dendritic mitochondria take up calcium (Ca2+) via the mitochondrial Ca2+ uniporter (MCU-1) that results in an upregulation of mitoROS production. We also observed that mitochondria are positioned in close proximity to synaptic clusters of GLR-1, the C. elegans ortholog of the AMPA subtype of glutamate receptors that mediate neuronal excitation. We show that synaptic recruitment of GLR-1 is upregulated when MCU-1 function is pharmacologically or genetically impaired but is downregulated by mitoROS signaling. Thus, signaling from postsynaptic mitochondria may regulate excitatory synapse function to maintain neuronal homeostasis by preventing excitotoxicity and energy depletion.

An operating principle of the cerebral cortex, and a cellular mechanism for attentional trial-and-error pattern learning and useful classification extraction.

A feature of the brains of intelligent animals is the ability to learn to respond to an ensemble of active neuronal inputs with a behaviorally appropriate ensemble of active neuronal outputs. Previously, a hypothesis was proposed on how this mechanism is implemented at the cellular level within the neocortical pyramidal neuron: the apical tuft or perisomatic inputs initiate "guess" neuron firings, while the basal dendrites identify input patterns based on excited synaptic clusters, with the cluster excitation strength adjusted based on reward feedback. This simple mechanism allows neurons to learn to classify their inputs in a surprisingly intelligent manner. Here, we revise and extend this hypothesis. We modify synaptic plasticity rules to align with behavioral time scale synaptic plasticity (BTSP) observed in hippocampal area CA1, making the framework more biophysically and behaviorally plausible. The neurons for the guess firings are selected in a voluntary manner via feedback connections to apical tufts in the neocortical layer 1, leading to dendritic Ca2+ spikes with burst firing, which are postulated to be neural correlates of attentional, aware processing. Once learned, the neuronal input classification is executed without voluntary or conscious control, enabling hierarchical incremental learning of classifications that is effective in our inherently classifiable world. In addition to voluntary, we propose that pyramidal neuron burst firing can be involuntary, also initiated via apical tuft inputs, drawing attention toward important cues such as novelty and noxious stimuli. We classify the excitations of neocortical pyramidal neurons into four categories based on their excitation pathway: attentional versus automatic and voluntary/acquired versus involuntary. Additionally, we hypothesize that dendrites within pyramidal neuron minicolumn bundles are coupled via depolarization cross-induction, enabling minicolumn functions such as the creation of powerful hierarchical "hyperneurons" and the internal representation of the external world. We suggest building blocks to extend the microcircuit theory to network-level processing, which, interestingly, yields variants resembling the artificial neural networks currently in use. On a more speculative note, we conjecture that principles of intelligence in universes governed by certain types of physical laws might resemble ours.