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Objective: Changes in the normal-appearing white matter (NAWM) in multiple sclerosis (MS) may contribute to disease progression. Here, we systematically quantified ultrastructural and subcellular characteristics of the axon–myelin unit in MS NAWM and determined how this correlates with low-grade inflammation. Methods: Human brain tissue obtained with short postmortem delay and fixation at autopsy enables systematic quantification of ultrastructural characteristics. In this study, we performed high-resolution immunohis tochemistry and quantitative transmission electron microscopy to study inflammation and ultrastructural characteristics of the axon–myelin unit in MS NAWM (n = 8) and control white matter (WM) in the optic nerve. Results: In the MS NAWM, there were more activated and phagocytic microglia cells (HLA +P2RY12 − and Iba1 +CD68 +) and more T cells (CD3 +) compared to control WM, mainly located in the perivascular space. In MS NAWM compared to control WM, there were, as expected, longer paranodes and juxtaparanodes and larger overlap between paranodes and juxtaparanodes. There was less compact myelin wrapping, a lower g-ratio, and a higher frequency of axonal mitochondria. Changes in myelin and axonal mitochondrial frequency correlated positively with the number of active and phagocytic microglia and lymphocytes in the optic nerve. Interpretation: These data suggest that in MS NAWM myelin detachment and uncompact myelin wrapping occurs, potassium channels are unmasked at the nodes of Ranvier, and axonal energy demand is increased, or mitochondrial transport is stagnated, accompanied by increased presence of activated and phagocytic microglia and T cells. These subclinical alterations to the axon–myelin unit in MS NAWM may contribute to disease progression. ANN NEUROL 2023;93:856–870.

Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients the genetic cause remains unknown. We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology. We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation. In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema.

Detailed characterization of interneuron subtypes in primary visual cortex (V1) has greatly contributed to understanding visual perception, yet the role of chandelier cells (ChCs) in visual processing remains poorly characterized. Using viral tracing we found that V1 ChCs predominantly receive monosynaptic input from local layer 5 pyramidal cells and higher-order cortical regions. Two-photon calcium imaging and convolutional neural network modelling revealed that ChCs are visually responsive but weakly selective for stimulus content. In mice running in a virtual tunnel, ChCs respond strongly to locomotion and halting visual flow, suggesting arousal-related activity. Visuomotor experience in the tunnel diminished visual responses of ChCs and induced structural plasticity of ChC boutons and axon initial segment length. Finally, ChCs only weakly inhibited pyramidal cells. These findings suggest that ChCs provide an arousal-related signal to layer 2/3 pyramidal cells that may modulate their activity and/or gate plasticity of their axon initial segments during behaviorally relevant events.

Changes in the normal-appearing white matter (NAWM) in multiple sclerosis (MS) may contribute to disease progression. Here, we systematically quantified ultrastructural and subcellular characteristics of the axon-myelin unit in MS NAWM and determined how this correlates with low grade inflammation.Human brain tissue obtained with short post-mortem delay and fixation at autopsy enables systematic quantification of ultrastructural characteristics. In this study, we performed high-resolution immunohistochemistry and quantitative transmission electron microscopy to study inflammation and ultrastructural characteristics of the axon-myelin unit in MS NAWM (n=8) and control white matter (WM) in the optic nerve.In the MS NAWM, there were more activated and phagocytic microglia cells (HLAThese data suggest that in MS NAWM myelin detachment and uncompact myelin wrapping occurs, potassium channels are unmasked at the nodes of Ranvier, and axonal energy demand is increased, or mitochondrial transport is stagnated, accompanied by increased presence of activated and phagocytic microglia and T cells. These sub-clinical alterations to the axon-myelin unit in MS NAWM may be contributing to disease progression. This article is protected by copyright. All rights reserved.

Human cortical pyramidal neurons are large, have extensive dendritic trees, and yet have surprisingly fast input-output properties: rapid subthreshold synaptic membrane potential changes are reliably encoded in timing of action potentials (APs). Here, we tested whether biophysical properties of voltage-gated sodium (Na+) and potassium (K+) currents in human neurons can explain their fast input-output properties. Human Na+and K+currents had depolarized voltage-dependence, slower inactivation and exhibited a faster recovery from inactivation than their mouse counterparts. Computational modeling showed that despite lower Na+channel densities in human neurons, the biophysical properties of Na+channels resulted in higher channel availability and explained fast AP kinetics stability. Finally, human Na+channel properties also resulted in a larger dynamic range for encoding of subthreshold membrane potential changes. Thus, biophysical adaptations of voltage-gated Na+and K+channels enable fast input-output properties of large human pyramidal neurons.One-Sentence SummaryBiophysical properties of Na+and K+ion channels enable human neurons to encode fast inputs into output.

Activity-dependent plasticity of the axon initial segment (AIS) endows neurons with the ability to adapt action potential output to changes in network activity. Action potential initiation at the AIS highly depends on the clustering of voltage-gated sodium channels, however the molecular mechanisms regulating their plasticity remain largely unknown. Here, we used novel genetic tools to endogenously label sodium channels and their scaffolding protein, to reveal their nanoscale organization and longitudinally image AIS plasticity in hippocampal neurons, in slices and primary cultures. We find that induction of NMDA receptor-mediated long-term synaptic depression is linked to a rapid and local endocytosis of sodium channels from the distal AIS. These data reveal a novel fundamental mechanism for rapid activity-dependent AIS reorganization sharing conserved features with synaptic plasticity.

SummaryParvalbumin-positive (PV+) γ-aminobutyric acid (GABA) interneurons are critically involved in producing rapid network oscillations and cortical microcircuit computations but the significance of PV+ axon myelination to the temporal features of inhibition remains elusive. Here using toxic and genetic models of demyelination and dysmyelination, respectively, we find that loss of compact myelin reduces PV+ interneuron presynaptic terminals, increases failures and the weak phasic inhibition of pyramidal neurons abolishes optogenetically driven gamma oscillations in vivo. Strikingly, during periods of quiet wakefulness selectively theta rhythms are amplified and accompanied by highly synchronized interictal epileptic discharges. In support of a causal role of impaired PV-mediated inhibition, optogenetic activation of myelin-deficient PV+ interneurons attenuated the power of slow theta rhythms and limited interictal spike occurrence. Thus, myelination of PV axons is required to consolidate fast inhibition of pyramidal neurons and enable behavioral state-dependent modulation of local circuit synchronization.

OBJECTIVE: In metachromatic leukodystrophy, a lysosomal storage disorder due to decreased arylsulfatase A activity, hematopoietic stem cell transplantation may stop brain demyelination and allow remyelination, thereby halting white matter degeneration. This is the first study to define the effects and therapeutic mechanisms of hematopoietic stem cell transplantation on brain tissue of transplanted metachromatic leukodystrophy patients.METHODS: Autopsy brain tissue was obtained from eight (two transplanted and six nontransplanted) metachromatic leukodystrophy patients, and two age-matched controls. We examined the presence of donor cells by immunohistochemistry and microscopy. In addition, we assessed myelin content, oligodendrocyte numbers, and macrophage phenotypes. An unpaired t-test, linear regression or the nonparametric Mann-Whitney U-test was performed to evaluate differences between the transplanted, nontransplanted, and control group.RESULTS: In brain tissue of transplanted patients, we found metabolically competent donor macrophages expressing arylsulfatase A distributed throughout the entire white matter. Compared to nontransplanted patients, these macrophages preferentially expressed markers of alternatively activated, anti-inflammatory cells that may support oligodendrocyte survival and differentiation. Additionally, transplanted patients showed higher numbers of oligodendrocytes and evidence for remyelination. Contrary to the current hypothesis on therapeutic mechanism of hematopoietic cell transplantation in metachromatic leukodystrophy, we detected no enzymatic cross-correction to resident astrocytes and oligodendrocytes.INTERPRETATION: In conclusion, donor macrophages are able to digest accumulated sulfatides and may play a neuroprotective role for resident oligodendrocytes, thereby enabling remyelination, albeit without evidence of cross-correction of oligo- and astroglia. These results emphasize the importance of immunomodulation in addition to the metabolic correction, which might be exploited for improved outcomes.

The complement system is implicated in synapse loss in the MS hippocampus, but the functional consequences of synapse loss remain poorly understood. Here, in post-mortem MS hippocampi with demyelination we find that deposits of the complement component C1q are enriched in the CA2 subfield, are linked to loss of inhibitory synapses and are significantly higher in MS patients with cognitive impairments compared to those with preserved cognitive functions. Using the cuprizone mouse model of demyelination, we corroborated that C1q deposits are highest within the demyelinated dorsal hippocampal CA2 pyramidal layer, and co-localized with inhibitory synapses engulfed by microglia/macrophages. In agreement with the loss of inhibitory perisomatic synapses, we further found that Schaffer collateral feedforward inhibition but not excitation was impaired in CA2 pyramidal neurons and accompanied by a reduced spike output. Ultimately, we show that these electrophysiological changes were associated with an impaired encoding of social memories. Together, our findings identify CA2 as a critical circuit in demyelinated intrahippocampal lesions and memory dysfunctions in MS.

Performing Single-Molecule Localization Microscopy (SMLM) in complex biological tissues, where sample-induced aberrations hamper detection and localization, has remained a challenge. Here we establish REALM (Robust and Effective Adaptive Optics in Localization Microscopy), which corrects aberrations of ≤1 rad RMS using 297 frames of blinking molecules to improve single-molecule localization. We demonstrate this method by resolving the periodic cytoskeleton of the axon initial segment at 50 μm depth in brain tissue.

Calcium ions (Ca2+) are essential for many cellular signaling mechanisms and enter the cytosol mostly through voltage-gated calcium channels. Here, using high-speed Ca2+imaging up to 20 kHz in the rat layer five pyramidal neuron axon we found that activity-dependent intracellular calcium concentration ([Ca2+]i) in the axonal initial segment was only partially dependent on voltage-gated calcium channels. Instead, [Ca2+]ichanges were sensitive to the specific voltage-gated sodium (NaV) channel blocker tetrodotoxin. Consistent with the conjecture that Ca2+enters through the NaVchannel pore, the optically resolvedICain the axon initial segment overlapped with the activation kinetics of NaVchannels and heterologous expression of NaV1.2 in HEK-293 cells revealed a tetrodotoxin-sensitive [Ca2+]irise. Finally, computational simulations predicted that axonal [Ca2+]itransients reflect a 0.4% Ca2+conductivity of NaVchannels. The findings indicate that Ca2+permeation through NaVchannels provides a submillisecond rapid entry route in NaV-enriched domains of mammalian axons.

SummaryThe axon initial segment (AIS) is an important axonal microdomain for action potential initiation and implicated in the regulation of neuronal excitability during activity-dependent cortical plasticity. While structural AIS plasticity has been suggested to fine-tune neuronal activity when network states change, whether it acts as a homeostatic regulatory mechanism in behaviorally relevant contexts remains poorly understood. Using an in vivo model of the mouse whisker-to-barrel pathway in combination with immunofluorescence, confocal analysis and patch-clamp electrophysiological recordings, we observed bidirectional AIS plasticity. Furthermore, we find that structural and functional AIS remodeling occurs in distinct temporal domains: long-term sensory deprivation elicits an AIS length increase, accompanied with an increase in neuronal excitability, while sensory enrichment results in a rapid AIS shortening, accompanied by a decrease in action potential generation. Our findings highlight a central role of the AIS in the homeostatic regulation of neuronal input-output relations.

OBJECTIVE: Loss of function of the astrocyte-specific protein MLC1 leads to the childhood-onset leukodystrophy "megalencephalic leukoencephalopathy with subcortical cysts" (MLC). Studies on isolated cells show a role for MLC1 in astrocyte volume regulation and suggest that disturbed brain ion and water homeostasis is central to the disease. Excitability of neuronal networks is particularly sensitive to ion and water homeostasis. In line with this, reports of seizures and epilepsy in MLC patients exist. However, systematic assessment and mechanistic understanding of seizures in MLC are lacking. METHODS: We analyzed an MLC patient inventory to study occurrence of seizures in MLC. We used two distinct genetic mouse models of MLC to further study epileptiform activity and seizure threshold through wireless extracellular field potential recordings. Whole-cell patch-clamp recordings and K+ -sensitive electrode recordings in mouse brain slices were used to explore the underlying mechanisms of epilepsy in MLC. RESULTS: An early onset of seizures is common in MLC. Similarly, in MLC mice, we uncovered spontaneous epileptiform brain activity and a lowered threshold for induced seizures. At the cellular level, we found that although passive and active properties of individual pyramidal neurons are unchanged, extracellular K+ dynamics and neuronal network activity are abnormal in MLC mice. INTERPRETATION: Disturbed astrocyte regulation of ion and water homeostasis in MLC causes hyperexcitability of neuronal networks and seizures. These findings suggest a role for defective astrocyte volume regulation in epilepsy. Ann Neurol 2018;83:636-649.

The complement system is implicated in synapse loss in the MS hippocampus, but the functional consequences of synapse loss remain poorly understood. Here, in post-mortem MS hippocampi with demyelination we find that deposits of the complement component C1q are enriched in the CA2 subfield, are linked to loss of inhibitory synapses and are significantly higher in MS patients with cognitive impairments compared to those with preserved cognitive functions. Using the cuprizone mouse model of demyelination, we corroborated that C1q deposits are highest within the demyelinated dorsal hippocampal CA2 pyramidal layer and co-localized with inhibitory synapses engulfed by microglia/macrophages. In agreement with the loss of inhibitory perisomatic synapses, we found that Schaffer collateral feedforward inhibition but not excitation was impaired in CA2 pyramidal neurons and accompanied by intrinsic changes and a reduced spike output. Finally, consistent with excitability deficits, we show that cuprizone-treated mice exhibit impaired encoding of social memories. Together, our findings identify CA2 as a critical circuit in demyelinated intrahippocampal lesions and memory dysfunctions in MS. Supplementary Information The online version contains supplementary material available at 10.1007/s00401-021-02338-8.

Myelinating oligodendrocytes enable fast propagation of action potentials along the ensheathed axons. In addition, oligodendrocytes play diverse non-canonical roles including axonal metabolic support and activity-dependent myelination. An open question remains whether myelination also contributes to information processing in addition to speeding up conduction velocity. Here, we analyze the role of myelin in auditory information processing using paradigms that are also good predictors of speech understanding in humans. We compare mice with different degrees of dysmyelination using acute multiunit recordings in the auditory cortex, in combination with behavioral readouts. We find complex alterations of neuronal responses that reflect fatigue and temporal acuity deficits. We observe partially discriminable but similar deficits in well myelinated mice in which glial cells cannot fully support axons metabolically. We suggest a model in which myelination contributes to sustained stimulus perception in temporally complex paradigms, with a role of metabolically active oligodendrocytes in cortical information processing., Oligodendrocytes myelinate and metabolically support axons. The role of myelination in information processing beyond regulation of conduction velocity is unclear. Here, the authors show that myelination contributes to sustained stimulus perception in the auditory cortex, shaping neuronal responses.

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels control electrical rhythmicity and excitability in the heart and brain, but the function of HCN channels at subcellular level in axons remains poorly understood. Here, we show that the action potential conduction velocity in both myelinated and unmyelinated central axons can bidirectionally be modulated by HCN channel blockers, cyclic adenosine monophosphate (cAMP), and neuromodulators. Recordings from mice cerebellar mossy fiber boutons show that HCN channels ensure reliable high-frequency firing and are strongly modulated by cAMP (EC50 40 µM; estimated endogenous cAMP concentration 13 µM). In accord, immunogold-electron microscopy revealed HCN2 as the dominating subunit in cerebellar mossy fibers. Computational modeling indicated that HCN2 channels control conduction velocity primarily via altering the resting membrane potential and was associated with significant metabolic costs. These results suggest that the cAMP-HCN pathway provides neuromodulators an opportunity to finely tune energy consumption and temporal delays across axons in the brain.

Myelinating oligodendrocytes enable fast impulse propagation along axons as revealed through studies of homogeneously myelinated white matter tracts. However, gray matter myelination patterns are different, with sparsely myelinated sections leaving large portions of the axons naked. The consequences of this patchy myelination for oligodendrocyte function are not understood but suggest other roles in information processing beyond the regulation of axonal conduction velocity. Here, we analyzed the contribution of myelin to auditory information processing using paradigms that are good predictors of speech understanding in humans. We compared mice with different degrees of dysmyelination using acute cortical multiunit recordings in combination with behavioral readouts. We identified complex alterations of neuronal responses that reflect fatigue and temporal acuity deficits. Partially discriminable but overall similar deficits were observed in mice with oligodendrocytes that can myelinate but cannot fully support axons metabolically. Thus, myelination contributes to sustained stimulus perception in temporally complex paradigms, revealing a role of oligodendrocytes in the CNS beyond the increase of axonal conduction velocity.

In mammalian neurons, the axon initial segment (AIS) electrically connects the somatodendritic compartment with the axon and converts the incoming synaptic voltage changes into a temporally precise action potential (AP) output code. Although axons often emanate directly from the soma, they may also originate more distally from a dendrite, the implications of which are not well-understood. Here, we show that one-third of the thick-tufted layer 5 pyramidal neurons have an axon originating from a dendrite and are characterized by a reduced dendritic complexity and thinner main apical dendrite. Unexpectedly, the rising phase of somatic APs is electrically indistinguishable between neurons with a somatic or a dendritic axon origin. Cable analysis of the neurons indicated that the axonal axial current is inversely proportional to the AIS distance, denoting the path length between the soma and the start of the AIS, and to produce invariant somatic APs, it must scale with the local somatodendritic capacitance. In agreement, AIS distance inversely correlates with the apical dendrite diameter, and model simulations confirmed that the covariation suffices to normalize the somatic AP waveform. Therefore, in pyramidal neurons, the AIS location is finely tuned with the somatodendritic capacitive load, serving as a homeostatic regulation of the somatic AP in the face of diverse neuronal morphologies.

The propagation of electrical impulses along axons is highly accelerated by the myelin sheath and produces saltating or "jumping" action potentials across internodes, from one node of Ranvier to the next. The underlying electrical circuit, as well as the existence and role of submyelin conduction in saltatory conduction remain, however, elusive. Here, we made patch-clamp and high-speed voltage-calibrated optical recordings of potentials across the nodal and internodal axolemma of myelinated neocortical pyramidal axons combined with electron microscopy and experimentally constrained cable modeling. Our results reveal a nanoscale yet conductive periaxonal space, incompletely sealed at the paranodes, which separates the potentials across the low-capacitance myelin sheath and internodal axolemma. The emerging double-cable model reproduces the recorded evolution of voltage waveforms across nodes and internodes, including rapid nodal potentials traveling in advance of attenuated waves in the internodal axolemma, revealing a mechanism for saltation across time and space.

While idiopathic generalized epilepsies are thought to evolve from temporal highly synchronized oscillations between thalamic and cortical networks, their cellular basis remains poorly understood. Here we show in a genetic rat model of absence epilepsy (WAG/Rij) that a rapid decline in expression of hyperpolarization-activated cyclic-nucleotide gated (HCN1) channels (I(h)) precedes the onset of seizures, suggesting that the loss of HCN1 channel expression is inherited rather than acquired. Loss of HCN1 occurs primarily in the apical dendrites of layer 5 pyramidal neurons in the cortex, leading to a spatially uniform 2-fold reduction in dendritic HCN current throughout the entire somato-dendritic axis. Dual whole-cell recordings from the soma and apical dendrites demonstrate that loss of HCN1 increases somato-dendritic coupling and significantly reduces the frequency threshold for generation of dendritic Ca2+ spikes by backpropagating action potentials. As a result of increased dendritic Ca2+ electrogenesis a large population of WAG/Rij layer 5 neurons showed intrinsic high-frequency burst firing. Using morphologically realistic models of layer 5 pyramidal neurons from control Wistar and WAG/Rij animals we show that the experimentally observed loss of dendritic I(h) recruits dendritic Ca2+ channels to amplify action potential-triggered dendritic Ca2+ spikes and increase burst firing. Thus, loss of function of dendritic HCN1 channels in layer 5 pyramidal neurons provides a somato-dendritic mechanism for increasing the synchronization of cortical output, and is therefore likely to play an important role in the generation of absence seizures.

The hyperpolarization-activated cation current (Ih) plays an important role in regulating neuronal excitability, yet its native single-channel properties in the brain are essentially unknown. Here we use variance-mean analysis to study the properties of singleIhchannels in the apical dendrites of cortical layer 5 pyramidal neuronsin vitro. In these neurons, we find thatIhchannels have an average unitary conductance of 680 ± 30 fS (n= 18). Spectral analysis of simulated and nativeIhchannels showed that there is little or no channel flicker below 5 kHz. In contrast to the uniformly distributed single-channel conductance,Ihchannel number increases exponentially with distance, reaching densities as high as ∼550 channels/μm2at distal dendritic sites. These high channel densities generate significant membrane voltage noise. By incorporating a stochastic model ofIhsingle-channel gating into a morphologically realistic model of a layer 5 neuron, we show that this channel noise is higher in distal dendritic compartments and increased threefold with a 10-fold increased single-channel conductance (6.8 pS) but constantIhcurrent density. In addition, we demonstrate that voltage fluctuations attributable to stochasticIhchannel gating impact on action potential output, with greater spike-timing precision in models with the experimentally determined single-channel conductance. These data suggest that, in the face of high current densities, the small single-channel conductance ofIhis critical for maintaining the fidelity of action potential output.

Neuronal subthreshold excitability and firing behaviour are markedly influenced by the activation and deactivation of the somato-dendritic hyperpolarization-activated cation current (Ih). Here, we evaluated possible contributions of Ih to hyperexcitability in an animal model of absence seizures (WAG/Rij rats). We investigated pyramidal neurons of the somatosensory neocortex, the site of generation of spike-wave discharges. Ih-mediated functions in neurons from WAG/Rij rats, Wistar rats (sharing the same genetic background with WAG/Rij, but less epilepsy-prone) and ACI rats (an inbred strain, virtually free of seizures) were compared. We complemented whole-cell recordings from layer 2-3 pyramidal neurons with immunohistochemistry, Western blot and RT-PCR analysis of the h-channel subunits HCN1-4. The fast component of Ih activation in WAG/Rij neurons was significantly reduced (50% reduction in the h-current density) and four times slower than in neurons from nonepileptic Wistar or ACI rats. The results showing decreases in currents corresponded to a 34% reduction in HCN1 protein in the WAG/Rij compared to the Wistar neocortex, but HCN1 mRNA showed stable expression. The other three Ih subunit mRNAs and proteins (HCN2-4) were not affected. The alterations in Ih magnitude and kinetics of gating in WAG/Rij neurons may contribute to augmented excitatory postsynaptic potentials, the increase in their temporal summation and the facilitation of burst firing of these neurons because each of these effects could be mimicked by the selective Ih antagonist ZD 7288. We suggest that the deficit in Ih-mediated functions may contribute to the development and onset of spontaneously occurring hyperexcitability in a rat model of absence seizures.

The negative impact of chronic stress at the structure of apical dendrite branches of cornu ammonis 3 (CA3) pyramidal neurons is well established. However, there is no information available on the CA3 dendritic organization related to short-lasting stress, which suffices to produce longterm habituation or sensitization of anxiety behaviors and neuroendocrine responses. Here, we tested the effects evoked by brief stress on the arrangements of CA3 pyramidal neuron dendrites, and the activity-dependent properties of the commissural-associational (C/A) excitatory postsynaptic potentials (EPSPs). Adult male rats were socially defeated followed by 3 weeks without further treatment or as comparison exposed to a regimen of a social defeat every second day for the same time period. We assessed CA3 pyramidal neurons with somatic whole-cell recording and neurobiotin application in acute hippocampal slices. The results from morphometric analysis of post hoc reconstructions demonstrated that CA3 dendrites from repeatedly stressed rats were reduced in surface area and length selectively at the apical cone (70% of control, approximately 280 mum from the soma). Brief stress, however, produced a similar decrease in apical dendritic length (77% of control, approximately 400 mum from the soma), accompanied by an increased length (167% of control) and branch complexity at the basal cone. The structural changes of the dendrites significantly influenced signal propagation by shortening the onset latency of EPSPs and increasing input resistance (r=0.45, P

The experience of chronic stress induces a reversible regression of hippocampal CA3 apical neuron dendrites. Although such postsynaptic membrane reduction will obviously diminish the possibility of synaptic input, the consequences for the functional membrane properties of these cells are not well understood. We tested the hypothesis that chronic stress affects the input-output characteristics and excitability of CA3 pyramidal cells. Somatic whole-cell current-clamp recording with parallel intracellular biocytin labeling was performed on CA3 neurons from in vitro hippocampal slices from male tree shrews, which were collected after 28 days of psychosocial stress exposure and compared to recordings obtained from control animals. Post hoc morphometric analysis of biocytin-labeled CA3 cells revealed branch regression, by fewer dendritic crossings and length, limited to a distance of similar to280-340 mum from the soma only. The results from whole-cell recording indicate that chronic stress surprisingly reduced the apparent membrane time constant and input resistance 20-25%, accompanied by increased amplitude of the hyperpolarization-induced voltage "sag." All active membrane properties, including depolarization-induced action potential kinetics, complex spiking patterns, and afterhyperpolarization voltages, were indistinguishable from control recordings. Although linear association analysis confirmed that differences in geometry, such as apical length or branch number, were correlated to functional variability in properties of the AP current and voltage threshold, these changes were too marginal to be reflected in the group differences. However, the individual adrenal hormone status was associated significantly with the selective changes in subthreshold excitability. Taken together, the data provide evidence that despite long-term stress induces morphological changes, upregulates cortisol release and shifts the intrinsic membrane properties, the efficacy of somatic excitability of CA3 pyramidal neurons is largely preserved. (C) 2004 Wiley-Liss, Inc.

Recent hypotheses on the action of antidepressants imply a modulation of excitatory amino acid transmission. Here, the effects of long-term antidepressant application in rats with the drug tianeptine were examined at hippocampal CA3 commissural associational (c/a) glutamate receptor ion channels, employing the whole-cell patch-clamp technique. The drug's impact was tested by subjecting rats to daily restraint stress for three weeks in combination with tianeptine treatment (10 mg/kg/day). Whereas stress increased the deactivation time-constant and amplitude of the N -methyl-d-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs), it did not affect the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptor-mediated EPSCs. Concomitant pharmacological treatment of stressed animals with tianeptine resulted in a normalized scaling of the amplitude ratio of NMDA receptor to AMPA/kainate receptor-mediated currents and prevented the stress-induced attenuation of NMDA-EPSCs deactivation. Both paired-pulse-facilitation and frequency-dependent plasticity remained unchanged. Both in control and stressed animals, however, tianeptine treatment strengthened the slope of the input-output relation of EPSCs. The latter was mimicked by exposing hippocampal slices in vitro with 10 mum tianeptine, which rapidly increased the amplitudes of NMDA- and AMPA/kainate EPSCs. The enhancement of EPSCs could be blocked by the intracellular presence of the kinase inhibitor staurosporine (1 mum), suggesting the involvement of a postsynaptic phosphorylation cascade rather then presynaptic release mechanisms at CA3 c/a synapses. These results indicate that tianeptine targets the phosphorylation-state of glutamate receptors at the CA3 c/a synapse. This novel signal transduction mechanism for tianeptine may provide a mechanistic resolution for its neuroprotective properties and, moreover, a pharmacological trajectory for its memory enhancing and/or antidepressant activity.

This study tested the time-of-day dependence of the intrinsic postsynaptic properties of hippocampal CA3 pyramidal neurons. High-voltage-activated Ca2+ currents and the Ca2+- and voltage-dependent afterhyperpolarizations were examined in slices of rat brains obtained at four distinct time periods. Just after onset of the dark phase, the steady-state amplitude of the Ca2+ current (-1.24 +/- 0.11 nA) was significantly greater (P

Action potential threshold is thought to be lowest in the axon, but when measured using conventional techniques, we found that action potential voltage threshold of rat cortical pyramidal neurons was higher in the axon than at other neuronal locations. In contrast, both current threshold and voltage threshold of the isolated somato-dendritic spike were substantially higher at the soma. These data indicate that action potential threshold is indeed lowest in the axon.

Central nervous system (CNS) inflammation involves the generation of inducible cytokines such as interferons (IFNs) and alterations in brain activity, yet the interplay of both is not well understood. Here, we show that in vivo elevation of IFNs by viral brain infection reduced hyperpolarization-activated currents (Ih) in cortical pyramidal neurons. In rodent brain slices directly exposed to type I IFNs, the hyperpolarization-activated cyclic nucleotide (HCN)-gated channel subunit HCN1 was specifically affected. The effect required an intact type I receptor (IFNAR) signaling cascade. Consistent with Ih inhibition, IFNs hyperpolarized the resting membrane potential, shifted the resonance frequency, and increased the membrane impedance. In vivo application of IFN-gamma to the rat and to the mouse cerebral cortex reduced the power of higher frequencies in the cortical electroencephalographic activity only in the presence of HCN1. In summary, these findings identify HCN1 channels as a novel neural target for type I IFNs providing the possibility to tune neural responses during the complex event of a CNS inflammation

Action potential generation and conduction requires large quantities of energy to restore Na + and K + ion gradients. We investigated the subcellular location and voltage dependence of this metabolic cost in rat neocortical pyramidal neurons. Using Na +K + charge overlap as a measure of action potential energy efficiency, we found that action potential initiation in the axon initial segment (AIS) and forward propagation into the axon were energetically inefficient, depending on the resting membrane potential. In contrast, action potential backpropagation into dendrites was efficient. Computer simulations predicted that, although the AIS and nodes of Ranvier had the highest metabolic cost per membrane area, action potential backpropagation into the dendrites and forward propagation into axon collaterals dominated energy consumption in cortical pyramidal neurons. Finally, we found that the high metabolic cost of action potential initiation and propagation down the axon is a trade-off between energy minimization and maximization of the conduction reliability of high-frequency action potentials. © 2012 Nature America, Inc. All rights reserved.

The axon initial segment (AIS) is a specialized membrane region in the axon of neurons where action potentials are initiated. Crucial to the function of the AIS is the presence of specific voltage-gated channels clustered at high densities, giving the AIS unique electrical properties. Here we review recent data on the physiology of the AIS. These data indicate that the role of the AIS is far richer than originally thought, leading to the idea that it represents a dynamic signal processing unit within neurons, regulating the integration of synaptic inputs, intrinsic excitability, and transmitter release. Furthermore, these observations point to a critical role of the AIS in disease.

In central neurons the first node of Ranvier is located at the first axonal branchpoint, similar to 100 mu m from the axon initial segment where synaptic inputs are integrated and converted into action potentials (APs). Whether the first node contributes to this signal transformation is not well understood. Here it was found that in neocortical layer 5 axons, the first branchpoint is required for intrinsic high-frequency (>= 100 Hz) AP bursts. Furthermore, block of nodal Na(+) channels or axotomy of the first node in intrinsically bursting neurons depolarized the somatic AP voltage threshold (similar to 5 mV) and eliminated APs selectively within a high-frequency cluster in response to steady currents or simulated synaptic inputs. These results indicate that nodal persistent Na+ current exerts an anterograde influence on AP initiation in the axon initial segment, revealing a computational role of the first node of Ranvier beyond conduction of the propagating AP

The axon initial segment (AIS) is a specialized region in neurons where action potentials are initiated. It is commonly assumed that this process requires a high density of voltage-gated sodium (Na(+)) channels. Paradoxically, the results of patch-clamp studies suggest that the Na(+) channel density at the AIS is similar to that at the soma and proximal dendrites. Here we provide data obtained by antibody staining, whole-cell voltage-clamp and Na(+) imaging, together with modeling, which indicate that the Na(+) channel density at the AIS of cortical pyramidal neurons is approximately 50 times that in the proximal dendrites. Anchoring of Na(+) channels to the cytoskeleton can explain this discrepancy, as disruption of the actin cytoskeleton increased the Na(+) current measured in patches from the AIS. Computational models required a high Na(+) channel density (approximately 2,500 pS microm(-2)) at the AIS to account for observations on action potential generation and backpropagation. In conclusion, action potential generation requires a high Na(+) channel density at the AIS, which is maintained by tight anchoring to the actin cytoskeleton.

The patch-clamp technique allows investigation of the electrical excitability of neurons and the functional properties and densities of ion channels. Most patch-clamp recordings from neurons have been made from the soma, the largest structure of individual neurons, while their dendrites, which form the majority of the surface area and receive most of the synaptic input, have been relatively neglected. This protocol describes techniques for recording from the dendrites of neurons in brain slices under direct visual control. Although the basic technique is similar to that used for somatic patching, we describe refinements and optimizations of slice quality, microscope optics, setup stability and electrode approach that are required for maximizing the success rate for dendritic recordings. Using this approach, all configurations of the patch-clamp technique (cell-attached, inside-out, whole-cell, outside-out and perforated patch) can be achieved, even for relatively distal dendrites, and simultaneous multiple-electrode dendritic recordings are also possible. The protocol--from the beginning of slice preparation to the end of the first successful recording--can be completed in 3 h.

In order to study mechanisms involved in the etiology of human affective disorders, there is an abundant use of various animal models. Next to genetic factors that predispose for psychopathologies, environmental stress is playing an important role in the etiology of these mental diseases. Since the majority of stress stimuli in humans that lead to psychopathology are of social nature, the study of consequences of social stress in experimental animal models is very valuable. The present review focuses on one of these models that uses the resident-intruder paradigm. In particular the long-lasting effects of social defeat in rats will be evaluated. Data from our laboratory on the consequences of social defeat on emotional behavior, stress responsivity and serotonergic functionality are presented. Furthermore, we will go into detail on hippocampal functioning in socially stressed rats. Very recent results show that there is a differential effect of a brief double social defeat and repetitive social defeat stress on dendritic remodeling in hippocampal CA3 neurons and that this has repercussions on hippocampal LTP and LTD. Both the structural and electrophysiological changes of principal neurons in the hippocampal formation after defeat are discussed as to their relationship with the maintenance in cognitive performance that was observed in socially stressed rats. The results are indicative of a large dynamic range in the adaptive plasticity of the brain, allowing the animals to adapt behaviorally to the previously occurred stressful situation with the progression of time.

Early hypotheses on the pathophysiology of major depression were based on aberrant intrasynaptic concentrations of mainly the neurotransmitters serotonin and norepinephrine. However, recent neuroimaging studies have demonstrated selective structural changes across various limbic and nonlimbic circuits in the brains of depressed patients. In addition, postmortem morphometric studies revealed decreased glial and neuron densities in selected brain structures supporting the idea that major depression may be related to impairments of structural plasticity. Stressful life events are among the major predisposing risk factors for developing depression. Using the chronic psychosocial stress paradigm in male tree shrews, an animal model with a high validity for the pathophysiology of depressive disorders, we found that 1 month of stress reduced the in vivo concentrations of the brain metabolites N-acetyl-aspartate, choline-containing compounds, and (phospho)-creatine, as well as the proliferation rate in the dentate gyrus and the hippocampal volume. Even though long-lasting social conflict does not lead to a loss of principal cells, the hippocampal changes were accompanied by modifications in the incidence of apoptosis. Notably, these suppressive effects of social conflict on hippocampal structure could be counteracted by treatment with the antidepressant tianeptine. These findings support current theories proposing that major depressive disorders may be associated with impairment of structural plasticity and neural cellular resilience, and that antidepressants may act by correcting this dysfunction.

Not long ago, glial cells were thought to have a typical linear rectification of membrane current and lack action potentials, a defining feature of neurons. But these boundaries are blurring. Oligodendrocytic precursor cells (OPCs), identified by expression of the proteoglycan NG2, appear during early postnatal life and maintain the capacity in adulthood to proliferate into oligodendrocytes producing myelin sheaths and performing myelin repair. OPCs appear to be a surprisingly excitable phenotype. They are morphologically characterized by radially projecting long and thin branches containing AMPA, NMDA and GABAergic receptors and receive direct synaptic inputs from neighbouring neurons (Lin & Bergles, 2004). Some NG2+ OPC classes even produce fast action potentials reminiscent of neurons (Karadottir et al. 2008). These findings raise the intriguing possibility that OPCs, similarly to neurons, perform intricate electrical computations. However, how synaptic currents spread along the OPC branches is not understood. In this issue of The Journal of Physiology, Chan et al. (2013) make a substantial contribution towards answering these emerging issues. Passive-cable theory, developed in the late 1950s by Rall and colleagues, provides a mathematical framework to describe the current flow and electrical characteristics of dendrites and axons by reducing these into equivalent cylinder models (Rall et al. 1992). The ability of a synapse, or its electrical voltage transient, to influence output can be analytically solved if one knows three basic parameters: the specific membrane resistance (Rm), intracellular core resistance (Ri) and membrane capacitance (Cm). These cable parameters, together with the specific cell geometry, determine the time course, spread and efficacy of current flow in neuronal dendrites. In a technical tour de force, Chan et al. used dual whole-cell patch-clamp recordings from rat hippocampal NG2+ OPCs combined with two-photon microscopy to create detailed three-dimensional anatomical reconstructions. Numerical compartmental models constrained by the morphology of the same cell were used to fit the recorded voltage transients and extract Rm, Ri and Cm (Rall et al. 1992). While Ri and Cm were within the range of values typically found in neurons, the authors show that OPCs have a unique low Rm. Remarkably, by placing the second recording electrode directly at one of the OPC processes, the specific membrane resistance was observed to distribute uniformly in the soma and branches. The development of an OPC passive-cable model creates important new insights. Firstly, information can be obtained from subcellular regions from which it is otherwise impossible to make recordings. For example, in the tip of the OPC branches simulated EPSPs were predicted to be larger in amplitude compared to the soma. Such non-uniformity in EPSP amplitudes can be explained by the time course of synaptic current, which in small and electrically isolated compartments can be fast enough to be independent of Rm, charging mainly the local input capacitance and intracellular resistance. Secondly, by virtue of a low Rm the EPSPs strongly attenuate in peak amplitude when propagating towards the soma, making the branches act as a linear filter. Thirdly, the EPSP amplitude and time course at the soma were found to be independent of synapse location, a phenomenon called ‘passive normalization’. The emerging picture of the OPC suggests a passive electrotonic structure that is optimized for highly compartmentalized input processing, a hypothesis that needs to be tested by, for example, imaging frequency-dependent Ca2+ responses in OPC processes. A low Rm reflects a high resting membrane conductance. Indeed, the authors identified that Rm is determined by inward-rectifying potassium (Kir) channels. Kir channels are known to be major players in glial physiology given their role in setting the resting membrane potential and regulation of extracellular uptake of K+. This is particularly interesting in light of the severe myelination deficits in Kir4.1 knockout mice (Neusch et al. 2001). Major new questions are arising from this study. Neurons use their cell-specific dendritic geometries and arrangement of biophysical membrane properties to enrich the computational capacity in the form of temporal coding with axonal action potential output. But what is the output of oligodendrocyte precursor glia? Is a sub-millisecond precise EPSP integration time window required and is there a temporal code to activate OPC proliferation and differentiation? This remains very speculative. NG2+ OPCs are efficiently recruited towards areas of myelin injury by means of the formation of new synaptic inputs (Etxeberria et al. 2010). Based on the passive-cable model, γ frequencies would be optimal for temporal summation in OPC branches. However, in vivo temporal summation will be much more complex than the passive scenario as OPCs also express voltage-gated sodium and calcium channels adding non-linearity to the membrane depolarization (Karadottir et al. 2008; Haberlandt et al. 2011). Future work needs to furnish the passive-cable models with active conductances to test whether OPC branches are capable of performing non-linear operations. In summary, the study of Chan et al. provides the basic tools for computational analyses of OPC functions. Compartmental models help interpret electrophysiological data but also make it possible to produce testable quantitative predictions, undoubtedly inspiring new electrophysiological and imaging studies to gain further insight into the rules of synaptic integration in oligodendrocyte precursors. Passively exciting new avenues in the study of glia physiology are therefore lying ahead.

Recent hypotheses on the action of antidepressants imply a modulation of excitatory amino acid transmission. Here, the effects of long-term antidepressant application in rats with the drug tianeptine were examined at hippocampal CA3 commissural associational (c/a) glutamate receptor ion channels, employing the whole-cell patch-clamp technique. The drug's impact was tested by subjecting rats to daily restraint stress for three weeks in combination with tianeptine treatment (10 mg/kg/day). Whereas stress increased the deactivation time-constant and amplitude of the N-methyl-d-aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs), it did not affect the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptor-mediated EPSCs. Concomitant pharmacological treatment of stressed animals with tianeptine resulted in a normalized scaling of the amplitude ratio of NMDA receptor to AMPA/kainate receptor-mediated currents and prevented the stress-induced attenuation of NMDA-EPSCs deactivation. Both paired-pulse-facilitation and frequency-dependent plasticity remained unchanged. Both in control and stressed animals, however, tianeptine treatment strengthened the slope of the input-output relation of EPSCs. The latter was mimicked by exposing hippocampal slices in vitro with 10 micro m tianeptine, which rapidly increased the amplitudes of NMDA- and AMPA/kainate EPSCs. The enhancement of EPSCs could be blocked by the intracellular presence of the kinase inhibitor staurosporine (1 micro m), suggesting the involvement of a postsynaptic phosphorylation cascade rather then presynaptic release mechanisms at CA3 c/a synapses. These results indicate that tianeptine targets the phosphorylation-state of glutamate receptors at the CA3 c/a synapse. This novel signal transduction mechanism for tianeptine may provide a mechanistic resolution for its neuroprotective properties and, moreover, a pharmacological trajectory for its memory enhancing and/or antidepressant activity.

Differential display of hippocampal tissue after entorhinal cortex lesion (ECL) revealed decreases in mRNA encoding the neuronal hyperpolarization-activated, cyclic nucleotide-gated channel HCN1. In situ hybridization confirmed that hippocampal transcripts of HCN1, but not HCN2/3/4, are down-regulated after ECL. Expression recovered at approximately 21 days after lesion (dal). Immunohistochemistry demonstrated a corresponding regulation of HCN1 protein expression in CA1-CA3 dendrites, hilar mossy cells and interneurons, and granule cells. Patch-clamp recordings in the early phase after lesion from mossy cells and hilar interneurons revealed an increase in the fast time constant of current activation and a profound negative shift in voltage activation of Ih. Whereas current activation recovered at 30 dal, the voltage activation remained hyperpolarized in mossy cells and hilar interneurons. Granule cells, however, were devoid of any detectable somatic Ih currents. Hence, denervation of the hippocampus decreases HCN1 and concomitantly the Ih activity in hilar neurons, and the recovery of h-current activation kinetics occurs parallel to postlesion sprouting.

The effects of a single rapid-rate transcranial magnetic stimulation (rTMS) exposure on neurotransmitter binding sites in the rat brain 24 h after the stimulation were examined. Quantification by in vitro-autoradiography showed no differences for H-3-paroxetine binding (5-HT uptake sites) between rTMS-treated, sham and control animals. In contrast, the number of 5-HT1A binding sites (labeled with H-3-8-OH-DPAT) were selectively increased in the rTMS-group with significantly higher B-MAX values in the frontal cortex, the cingulate cortex, and the anterior olfactory nucleus. A non-specific increase in NMDA binding sites (labeled with I-125-MK-801) in rTMS and sham animals was observed in the hippocampal formation. A selective increase of these binding sites after rTMS was detected in the ventromedial hypothalamus, the basolateral amygdala and layers 5-6 of the parietal cortex. These findings imply that a single rTMS exposure can result in persistent effects on NMDA and 5-HT1A binding sites even 24 h after stimulation and therefore may be of relevance with respect to the therapeutic action of rTMS reported from clinical studies. (C) 1999 Elsevier Science B.V. All rights reserved.

SummaryAction potentials are binary signals that transmit information via their rate and temporal pattern. In this context, the axon is thought of as a transmission line, devoid of a role in neuronal computation. Here, we show a highly localized role of axonal Kv1 potassium channels in shaping the action potential waveform in the axon initial segment (AIS) of layer 5 pyramidal neurons independent of the soma. Cell-attached recordings revealed a 10-fold increase in Kv1 channel density over the first 50 μm of the AIS. Inactivation of AIS and proximal axonal Kv1 channels, as occurs during slow subthreshold somatodendritic depolarizations, led to a distance-dependent broadening of axonal action potentials, as well as an increase in synaptic strength at proximal axonal terminals. Thus, Kv1 channels are strategically positioned to integrate slow subthreshold signals, providing control of the presynaptic action potential waveform and synaptic coupling in local cortical circuits.