Zoltan Nusser

The hippocampal CA1 area is perceived as a nonrecurrent neuronal network, i.e., CA1 pyramidal cells (CA1PCs) within the CA1 area innervate mainly γ-aminobutyric acid–releasing interneurons but not each other. Here, we performed an unbiased examination of the functional properties of local CA1PC output synapses using in vitro two-photon glutamate imaging. Fluorescent transients measured with iGluSnFR3 in individual boutons displayed extraordinary heterogeneity in their amplitudes and short-term plasticity patterns upon a complex train of presynaptic action potential firing. Post hoc electron microscopy of the imaged boutons demonstrated that distinct postsynaptic structure-innervating boutons display different functional properties. Approximately 25% of the boutons innervate CA1PC spines, which either release glutamate with very low release probability or completely fail to release. Phorbol 12,13-dibutyrate and elevated calcium (2+) concentration, manipulations that enhance synaptic vesicle priming and fusion, convert these silent into properly functioning synapses. Our results provide evidence that CA1PCs are interconnected with structurally and molecularly intact glutamatergic synapses, but incomplete synaptic vesicle (SV) priming precludes SV release.

Synaptic vesicle (SV) release probability ( Pv ) is determined by two probabilistic factors: the probability of release sites being occupied by fusion-competent, well-primed SVs and their fusion probability ( P _fusion ). While recent studies emphasize SV priming as a key mechanism underlying functional synaptic diversity, disentangling priming from fusion is notoriously challenging. Here we developed a mouse genetic approach for inducible and selective increase of SV priming. A histidine-to-lysine mutation at position 567 of Munc13-1 increases its function. Combining this mutation with a Cre-dependent removal of the wild-type Munc13-1 allele enables cell type–selective enhancement of Munc13-1 function. This manipulation increased excitatory postsynaptic current amplitude at hippocampal synapses exclusively through elevating Pv without affecting release site number or quantal size. A sequential, two-step priming model predicts that the enhanced Pv results from an elevated proportion of well-primed SVs, without altering P _fusion . Last, we provide unequivocal evidence that the postsynaptic target cell type–dependent variability in presynaptic glutamate release is mainly the consequence of variability in SV priming.

Every explored environment is represented in the hippocampus by the activity of distinct populations of pyramidal cells (PCs) that typically fire at specific locations called their place fields (PFs). New PFs are constantly born even in familiar surroundings (during representational drift), and many rapidly emerge when the animal explores a new or altered environment (during global or partial remapping). Behavioral time scale synaptic plasticity (BTSP), a plasticity mechanism based on prolonged somatic action potential (AP) bursts induced by dendritic Ca ²⁺ /NMDA plateau potentials, was recently proposed as the main cellular mechanism underlying new PF formations (PFFs), but it is unclear whether burst-associated large somatic [Ca ²⁺ ] transients are always necessary and/or sufficient for PFF. To address this issue, here we performed in vivo two-photon [Ca ²⁺ ] imaging of hippocampal CA1 PCs in head-restrained mice to investigate somatic [Ca ²⁺ ] dynamics underlying PFFs in familiar and novel virtual environments. Our results demonstrate that although many PFs are formed by BTSP-like events, PFs also emerge with initial [Ca ²⁺ ] dynamics that do not match any of the characteristics of BTSP. BTSP- and non-BTSP-like new PFFs occur spontaneously in familiar environments, during neuronal representational switches, and instantaneously in new environments. Our data also reveal that solitary [Ca ²⁺ ] transients with larger amplitudes than those evoking BTSP-like PFFs frequently occur without inducing PFs, demonstrating that large [Ca ²⁺ ] transients per se are not sufficient for PFF.

Every explored environment is represented in the hippocampus by the activity of distinct populations of pyramidal cells (PCs) that typically fire at specific locations called their place fields (PFs). New PFs are constantly born even in familiar surroundings (during representational drift), and many rapidly emerge when the animal explores a new or altered environment (during global or partial remapping). Behavioral time scale synaptic plasticity (BTSP), a plasticity mechanism based on prolonged somatic action potential (AP) bursts induced by dendritic Ca2+/NMDA plateau potentials, was recently proposed as the main cellular mechanism underlying new PF formations (PFF), but it is unclear whether burst-associated large somatic [Ca2+] transients are always necessary and/or sufficient for PFF. To address this issue, here we performed in vivo two-photon [Ca2+] imaging of hippocampal CA1 PCs in head-restrained mice to investigate somatic [Ca2+] dynamics underlying PFFs in familiar and novel virtual environments. Our results demonstrate that although many PFs are formed by BTSP-like events, PFs also emerge with initial [Ca2+] dynamics that do not match any of the characteristics of BTSP. BTSP and non-BTSP-like new PFFs occur spontaneously in familiar environments, during neuronal representational switches and instantaneously in new environments. Our data also reveal that solitary [Ca2+] transients with larger amplitudes than those evoking BTSP-like PFFs, frequently occur without inducing PFs, demonstrating that large [Ca2+] transients per se are not sufficient for PFF.

Abnormal excitatory synaptic transmission in the human prefrontal cortex has been implicated in the pathophysiology of schizophrenia based primarily on genetic evidence. However, changes in synaptic function cannot be predicted from altered gene expressions, but determining the amount, density, and subsynaptic distribution of synaptic proteins is the only reliable indirect readout of function. Detecting proteins in individual synapses of human postmortem tissues has been severely constrained by technical limitations. Here we overcome this limitation by optimizing a high-resolution, quantitative localization method to facilitate antigen recognition at excitatory synapses in postmortem brains of both sexes. Using PSD-95 immunoreactivity as molecular marker of excitatory synapses, we demonstrate the lack of significant differences in synapse density and size in upper cortical layers of control and schizophrenia subjects. The synaptic densities of postsynaptic AMPA and NMDA receptor subunits and presynaptic molecules Bassoon and Munc13-1 are also indistinguishable between control and schizophrenia subjects. The number of Munc13-1 nanoclusters, marking presynaptic neurotransmitter release sites, does not differ either. Excitatory synapses on parvalbumin expressing interneurons contain similar AMPA but significantly lower NMDA receptor densities in schizophrenia compared with control subjects. Our study provides the first comprehensive comparison of key functionally relevant synaptic proteins in individual human excitatory synapses and demonstrates that changes in the molecular composition of only a specific subset of excitatory synapses may contribute to the pathophysiology of schizophrenia.

Neuronal activity in the hippocampus creates a cognitive map of space that is essential for navigation. In any given environment, a fraction of hippocampal pyramidal cells (PCs) is active at specific locations (place cells), others are sparsely active without spatial tuning, and a significant proportion of the PCs is entirely silent. The mechanisms underlying the vastly different activities of PCs in the rodent hippocampal CA1 area are unknown. Here, we measured the in vivo activity of CA1 PCs using two-photon [Ca ²⁺ ] imaging in head-restrained mice during navigation in a virtual corridor and then performed in vitro patch-clamp recording to probe their intrinsic electrical properties and anatomical investigation to characterize their input synapses. The active and passive electrical properties of PCs were similar between PCs with different prior in vivo activities. Perisomatic inhibitory synapse density was also comparable among PCs. The average dendritic spine density and spine head area did not correlate with the mean in vivo activity of PCs, but the size of the spines of place cells was significantly larger compared to that of silent cells. Our results are consistent with excitatory synaptic plasticity as a major mechanism underlying spatially tuned activity of place cells in hippocampal networks.

Every explored environment is represented in the hippocampus by the activity of distinct populations of pyramidal cells (PCs) that typically fire at specific locations called their place fields (PFs). New PFs are constantly born even in familiar surroundings (during representational drift), and many rapidly emerge when the animal explores a new or altered environment (during global or partial remapping). Behavioral time scale synaptic plasticity (BTSP), a plasticity mechanism based on prolonged somatic action potential (AP) bursts induced by dendritic Ca ²⁺ /NMDA plateau potentials, was recently proposed as the main cellular mechanism underlying new PF formations (PFF), but it is unclear whether burst-associated large somatic [Ca ²⁺ ] transients are always necessary and/or sufficient for PFF. To address this issue, here we performed in vivo two-photon [Ca ²⁺ ] imaging of hippocampal CA1 PCs in head-restrained mice to investigate somatic [Ca ²⁺ ] dynamics underlying PFFs in familiar and novel virtual environments. Our results demonstrate that although many PFs are formed by BTSP-like events, PFs also emerge with initial [Ca ²⁺ ] dynamics that do not match any of the characteristics of BTSP. BTSP and non-BTSP-like new PFFs occur spontaneously in familiar environments, during neuronal representational switches and instantaneously in new environments. Our data also reveal that solitary [Ca ²⁺ ] transients with larger amplitudes than those evoking BTSP-like PFFs, frequently occur without inducing PFs, demonstrating that large [Ca ²⁺ ] transients per se are not sufficient for PFF.

Unsupervised segmentation in biological and non-biological images is only partially resolved. Segmentation either requires arbitrary thresholds or large teaching datasets. Here we propose a spatial autocorrelation method based on Local Moran’s I coefficient to differentiate signal, background and noise in any type of image. The method, originally described for geoinformatics, does not require a predefined intensity threshold or teaching algorithm for image segmentation and allows quantitative comparison of samples obtained in different conditions. It utilizes relative intensity as well as spatial information of neighboring elements to select spatially contiguous groups of pixels. We demonstrate that Moran’s method outperforms threshold-based method (TBM) in both artificially generated as well as in natural images especially when background noise is substantial. This superior performance can be attributed to the exclusion of false positive pixels resulting from isolated, high intensity pixels in high noise conditions. To test the method’s power in real situation we used high power confocal images of the somatosensory thalamus immunostained for Kv4.2 and Kv4.3 (A-type) voltage gated potassium channels. Moran’s method identified high intensity Kv4.2 and Kv4.3 ion channel clusters in the thalamic neuropil. Spatial distribution of these clusters displayed strong correlation with large sensory axon terminals of subcortical origin. The unique association of the special presynaptic terminals and a postsynaptic voltage gated ion channel cluster was confirmed with electron microscopy. These data demonstrate that Moran’s method is a rapid, simple image segmentation method optimal for variable and high nose conditions.

Pronounced differences in neurotransmitter release from a given presynaptic neuron, depending on the synaptic target, are among the most intriguing features of cortical networks. Hippocampal pyramidal cells (PCs) release glutamate with low probability to somatostatin expressing oriens-lacunosum-moleculare (O-LM) interneurons (INs), and the postsynaptic responses show robust short-term facilitation, whereas the release from the same presynaptic axons onto fast-spiking INs (FSINs) is ~10-fold higher and the excitatory postsynaptic currents (EPSCs) display depression. The mechanisms underlying these vastly different synaptic behaviors have not been conclusively identified. Here, we applied a combined functional, pharmacological, and modeling approach to address whether the main difference lies in the action potential-evoked fusion or else in upstream priming processes of synaptic vesicles (SVs). A sequential two-step SV priming model was fitted to the peak amplitudes of unitary EPSCs recorded in response to complex trains of presynaptic stimuli in acute hippocampal slices of adult mice. At PC–FSIN connections, the fusion probability (P _fusion ) of well-primed SVs is 0.6, and 44% of docked SVs are in a fusion-competent state. At PC–O-LM synapses, P _fusion is only 40% lower (0.36), whereas the fraction of well-primed SVs is 6.5-fold smaller. Pharmacological enhancement of fusion by 4-AP and priming by PDBU was recaptured by the model with a selective increase of P _fusion and the fraction of well-primed SVs, respectively. Our results demonstrate that the low fidelity of transmission at PC–O-LM synapses can be explained by a low occupancy of the release sites by well-primed SVs.

Unsupervised segmentation in biological and non-biological images is only partially resolved. Segmentation either requires arbitrary thresholds or large teaching datasets. Here we propose a spatial autocorrelation method based on Local Moran's I coefficient to differentiate signal, background and noise in any type of image. The method, originally described for geoinformatics, does not require a predefined intensity threshold or teaching algorithm for image segmentation and allows quantitative comparison of samples obtained in different conditions. It utilizes relative intensity as well as spatial information of neighboring elements to select spatially contiguous groups of pixels. We demonstrate that Moran's method outperforms threshold-based method (TBM) in both artificially generated as well as in natural images especially when background noise is substantial. This superior performance can be attributed to the exclusion of false positive pixels resulting from isolated, high intensity pixels in high noise conditions. To test the method's power in real situation we used high power confocal images of the somatosensory thalamus immunostained for Kv4.2 and Kv4.3 (A-type) voltage gated potassium channels. Moran's method identified high intensity Kv4.2 and Kv4.3 ion channel clusters in the thalamic neuropil. Spatial distribution of these clusters displayed strong correlation with large sensory axon terminals of subcortical origin. The unique association of the special presynaptic terminals and a postsynaptic voltage gated ion channel cluster was confirmed with electron microscopy. These data demonstrate that Moran's method is a rapid, simple image segmentation method optimal for variable and high nose conditions.