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The roles GABAergic and glutamatergic inputs in regulating the activity of the gonadotrophin-releasing hormone (GnRH) neurons at the time of the preovulatory surge remain unclear. We used expansion microscopy to compare the density of GABAergic and glutamatergic synapses on the GnRH neuron cell body and proximal dendrite in dioestrous and pro-oestrous female mice. An evaluation of all synapses immunoreactive for synaptophysin revealed that the highest density of inputs to rostral preoptic area GnRH neurons occurred within the first 45 µm of the primary dendrite (approximately 0.19 synapses µm-1 ) with relatively few synapses on the GnRH neuron soma or beyond 45 µm of the dendrite (0.05-0.08 synapses µm-1 ). Triple immunofluorescence labelling demonstrated a predominance of glutamatergic signalling with twice as many vesicular glutamate transporter 2 synapses detected compared to vesicular GABA transporter. Co-labelling with the GABAA receptor scaffold protein gephyrin and the glutamate receptor postsynaptic density marker Homer1 confirmed these observations, as well as the different spatial distribution of GABA and glutamate inputs along the dendrite. Quantitative assessments revealed no differences in synaptophysin, GABA or glutamate synapses at the proximal dendrite and soma of GnRH neurons between dioestrous and pro-oestrous mice. Taken together, these studies demonstrate that the GnRH neuron receives twice as many glutamatergic synapses compared to GABAergic synapses and that these inputs preferentially target the first 45 µm of the GnRH neuron proximal dendrite. These inputs appear to be structurally stable before the onset of pro-oestrous GnRH surge.

Among all voltage-gated potassium (Kv) channels, Kv2 channels are the most widely expressed in the mammalian brain. However, studying Kv2 in neurons has been challenging due to a lack of high-selective blockers. Recently, a peptide toxin, guangxitoxin-1E, has been identified as a specific inhibitor of Kv2, thus facilitating the study of Kv2 in neurons. The mammalian dorsal cochlear nucleus (DCN) integrates auditory and somatosensory information. In the DCN, cartwheel inhibitory interneurons receive excitatory synaptic inputs from parallel fibers conveying somatosensory information. The activation of parallel fibers drives action potentials in the cartwheel cells up to 130 Hz in vivo, and the excitation of cartwheel cells leads to the strong inhibition of principal cells. Therefore, cartwheel cells play crucial roles in monaural sound localization and cancelling detection of self-generated sounds. However, how Kv2 controls the high-frequency firing in cartwheel cells is unknown. In this study, we performed immunofluorescence labeling with anti-Kv2.1 and anti-Kv2.2 antibodies using fixed mouse brainstem slice preparations. The results revealed that Kv2.1 and Kv2.2 were largely present on the cartwheel cell body membrane but not on the axon initial segment nor the proximal dendrite. Whole-cell patch clamp recordings using mouse brainstem slice preparation and guangxitoxin-1E demonstrated that blockade of Kv2 induced failure of parallel fiber-induced action potentials when parallel fibers were stimulated at high frequencies (30-100 Hz). Thus, somatic Kv2 in cartwheel cells regulates the action potentials in a frequency-dependent manner and may play important roles in the DCN function. Significance statement The mammalian dorsal cochlear nucleus (DCN) plays a role in the monaural sound localization and cancelling detection of self-generated sounds. In the DCN, cartwheel cells receive excitatory synaptic inputs from parallel fibers. Parallel fiber activation can drive action potentials in cartwheel cells at high frequency, but the ionic mechanism of such firing remains unknown. In this study, we found that Kv2.1 and Kv2.2 ion channels were present on the cell bodies of cartwheel cells. Application of a specific blocker of Kv2 induced failure of parallel fiber-induced action potentials only when presynaptic parallel fibers were stimulated at high frequencies. Thus, somatic Kv2 in cartwheel cells regulates action potentials in a frequency-dependent manner and may play important roles in sound processing.

Key points Neurons of the lateral superior olive (LSO) in the brainstem receive powerful glycinergic inhibition that originates from the contralateral ear, and that plays an important role in sound localization. We investigated the ultrastructural basis for strong inhibition of LSO neurons using serial block face scanning electron microscopy. The soma and the proximal dendrite of an LSO neuron are surrounded by a high density of inhibitory axons, whereas excitatory axons are much sparser. A given inhibitory axon establishes contacts via several large axonal thickenings, called varicosities, which typically elaborate several active zones (range 1-11). The number of active zones across inhibitory axon segments is variable. These data thus provide an ultrastructural correlate for the strong and multiquantal, but overall variable, unitary IPSC amplitude observed for inhibitory inputs to LSO neuron. Abstract Binaural neurons in the lateral superior olive (LSO) integrate sound information arriving from each ear, and powerful glycinergic inhibition of these neurons plays an important role in this process. In the present study, we investigated the ultrastructural basis for strong inhibitory inputs onto LSO neurons using serial block face scanning electron microscopy. We reconstructed axon segments that make contact with the partially reconstructed soma and proximal dendrite of a mouse LSO neuron at postnatal day 18. Using functional measurements and the Sr2+ method, we find a constant quantal size but a variable quantal content between 'weak' and 'strong' unitary IPSCs. A 3-D reconstruction of a LSO neuron and its somatic synaptic afferents reveals how a large number of inhibitory axons intermingle in a complex fashion on the soma and proximal dendrite of an LSO neuron; a smaller number of excitatory axons was also observed. A given inhibitory axon typically contacts an LSO neuron via several large varicosities (average diameter 3.7 μm), which contain several active zones (range 1-11). The number of active zones across individual axon segments was highly variable. These data suggest that the variable unitary IPSC amplitude is caused by a variable number of active zones between inhibitory axons that innervate a given LSO neuron. The results of the present study show that relatively large multi-active zone varicosities, which can be repeated many times in a given presynaptic axon, provide the ultrastructural basis for the strong multiquantal inhibition received by LSO neurons.

In this paper, we proposed an extended version of binary code selection algorithm (BCSA) for the variable-order prediction by introducing multiple distal dendrites into BCSA. The proposed model of artificial neurons has a single proximal dendrite to receive the feed-forward inputs (sequences) from the world and multiple distal dendrites to receive the horizontal inputs from nearby neurons. During training, each distal dendrite is able to remember the states of neurons activated at different time and store the temporal correlations. After training, each distal dendrite independently recalls the temporal correlations contained in sequences and makes a local prediction. The variable-order prediction can be obtained by combining these local predictions made by multiple distal dendrites. Experiments show that the proposed method outperforms BCSA and other methods, such as back-propagation networks and radial basis function networks, especially while processing complex sequences.

Neuroscience Hippocampal neurons receive and integrate synaptic input along their dendritic tree. Inputs located near the cell soma or in the distal dendrite contribute differently to neuronal integration by a variety of mechanisms, including NMDA receptor (NMDAR)–dependent plasticity processes. Using superresolution microscopy, single-nanoparticle imaging, and glutamate uncaging, Ferreira et al. investigated the nanoscale organization of NMDARs containing the subunits GluN2A and GluN2B along the dendritic tree. The organization and surface dynamics of GluN2B-NMDARs, but not of GluN2A-NMDARs, changed between proximal and distal clusters, with a gradual increase in receptor local density from proximal to distal dendritic segments. At the proximal dendrite, the nanoscale organization and membrane dynamics of GluN2B-NMDARs were influenced by physical interplay with the protein kinase CaMKII. Proc. Natl. Acad. Sci. U.S.A. 117 , 24526 (2020).

Corticosteroid stress hormones drive a multitude of adaptations in the brain. Hypothalamic corticotropin-releasing hormone (CRH) neurons control the circulating levels of corticosteroid stress hormones in the body and are themselves highly sensitive to corticosteroids. CRH neurons have been shown to undergo various adaptions in response to acute stress hormone elevations. However, their structural and physiological changes under chronically elevated corticosterone are less clear. To address this, we determined the structural and functional changes in CRH neurons in the paraventricular nucleus of the hypothalamus following 14 days of corticosterone treatment. We find that prolonged corticosterone elevation reduces CRH neuron intrinsic excitability as measured by summation of subthreshold postsynaptic depolarisations and spiking output. We find that under normal conditions, CRH neurons have a relatively compact and simple dendritic arbor, with a low density of somatic and dendritic spines. Interestingly, the axon originated from a proximal dendrite close to the soma in approximately half of the CRH neurons reconstructed. While prolonged elevation in corticosterone levels did not result in any changes to gross dendritic morphology, it induced a significant reduction in both somatic and dendritic spine density. Together these data reveal the morphological features of hypothalamic CRH neurons and highlight their capacity to undergo functional and morphological plasticity in response to chronic corticosterone elevations. This article is part of the Special Issue entitled 'Hypothalamic Control of Homeostasis'.

GnRH neurons are the final output neurons of the hypothalamic network controlling fertility in mammals. In the present study, we used ankyrin G immunohistochemistry and neurobiotin filling of live GnRH neurons in brain slices from GnRH-green fluorescent protein transgenic male mice to examine in detail the location of action potential initiation in GnRH neurons with somata residing at different locations in the basal forebrain. We found that the vast majority of GnRH neurons are bipolar in morphology, elaborating a thick (primary) and thinner (secondary) dendrite from opposite poles of the soma. In addition, an axon-like process arising predominantly from a proximal dendrite was observed in a subpopulation of GnRH neurons. Ankyrin G immunohistochemistry revealed the presence of a single action potential initiation zone ∼27 μm in length primarily in the secondary dendrite of GnRH neurons and located 30 to 140 μm distant from the cell soma, depending on the type of process and location of the cell body. In addition to dendrites, the GnRH neurons with cell bodies located close to hypothalamic circumventricular organs often elaborated ankyrin G–positive axon-like structures. Almost all GnRH neurons (>90%) had their action potential initiation site in a process that initially, or ultimately after a hairpin loop, was coursing in the direction of the median eminence. These studies indicate that action potentials are initiated in different dendritic and axonal compartments of the GnRH neuron in a manner that is dependent partly on the neuroanatomical location of the cell body.

Neurones in the arcuate nucleus that express neurokinin B (NKB), kisspeptin and dynorphin (KNDy) play an important role in the reproductive axis. Oestradiol modulates the gene expression and somatic size of these neurones, although there is limited information available about whether their dendritic structure, a correlate of cellular plasticity, is altered by oestrogens. In the present study, we investigated the morphology of KNDy neurones by filling fluorescent neurones in the arcuate nucleus of Tac2-enhanced green fluorescent protein (EGFP) transgenic mice with biocytin. Filled neurones from ovariectomised (OVX) or OVX plus 17β-oestradiol (E2)-treated mice were visualised with anti-biotin immunohistochemistry and reconstructed in three dimensions with computer-assisted microscopy. KNDy neurones exhibited two primary dendrites, each with a few branches confined to the arcuate nucleus. Quantitative analysis revealed that E2 treatment of OVX mice decreased the cell size and dendritic spine density of KNDy neurones. The axons of KNDy neurones originated from the cell body or proximal dendrite and gave rise to local branches that appeared to terminate within the arcuate nucleus. Numerous terminal boutons were also visualised within the ependymal layer of the third ventricle adjacent to the arcuate nucleus. Axonal branches also projected to the adjacent median eminence and exited the arcuate nucleus. Confocal microscopy revealed close apposition of EGFP and gonadotrophin-releasing hormone-immunoreactive fibres within the median eminence and confirmed the presence of KNDy axon terminals in the ependymal layer of the third ventricle. The axonal branching pattern of KNDy neurones suggests that a single KNDy neurone could influence multiple arcuate neurones, tanycytes in the wall of the third ventricle, axon terminals in the median eminence and numerous areas outside of the arcuate nucleus. In parallel with its inhibitory effects on electrical excitability, E2 treatment of OVX Tac2-EGFP mice induces structural changes in the somata and dendrites of KNDy neurones.

In Purkinje cells (PCs) of the cerebellum, a single “winner” climbing fiber (CF) monopolizes proximal dendrites, whereas hundreds of thousands of parallel fibers (PFs) innervate distal dendrites, and both CF and PF inputs innervate a narrow intermediate domain. It is unclear how this segregated CF and PF innervation is established on PC dendrites. Through reconstruction of dendritic innervation by serial electron microscopy, we show that from postnatal day 9–15 in mice, both CF and PF innervation territories vigorously expand because of an enlargement of the region of overlapping innervation. From postnatal day 15 onwards, segregation of these territories occurs with robust shortening of the overlapping proximal region. Thus, innervation territories by the heterologous inputs are refined during the early postnatal period. Intriguingly, this transition is arrested in mutant mice lacking the type 1 metabotropic glutamate receptor (mGluR1) or protein kinase Cγ (PKCγ), resulting in the persistence of an abnormally expanded overlapping region. This arrested territory refinement is rescued by lentivirus-mediated expression of mGluR1α into mGluR1-deficient PCs. At the proximal dendrite of rescued PCs, PF synapses are eliminated and free spines emerge instead, whereas the number and density of CF synapses are unchanged. Because the mGluR1-PKCγ signaling pathway is also essential for the late-phase of CF synapse elimination, this signaling pathway promotes the two key features of excitatory synaptic wiring in PCs, namely CF monoinnervation by eliminating redundant CF synapses from the soma, and segregated territories of CF and PF innervation by eliminating competing PF synapses from proximal dendrites.

T-type calcium channels play a pivotal role in regulating neural membrane excitability in the nervous system. However, the precise subcellular distributions of T-type channel subunits and their implication for membrane excitability are not well understood. Here we investigated the subcellular distribution of the α1G subunit of the calcium channel which is expressed highly in the mouse dorsal lateral geniculate nucleus (dLGN). Light microscopic analysis demonstrated that dLGN exhibits intense immunoperoxidase reactivity for the α1G subunit. Electron microscopic observation showed that the labeling was present in both the relay cells and interneurons and was found in the somatodendritic, but not axonal, domains of these cells. Most of the immunogold particles for the α1G subunit were either associated with the plasma membrane or the intracellular membranes. Reconstruction analysis of serial electron microscopic images revealed that the intensity of the intracellular labeling exhibited a gradient such that the labeling density was higher in the proximal dendrite and progressively decreased towards the distal dendrite. In contrast, the plasma membrane-associated particles were distributed with a uniform density over the somatodendritic surface of dLGN cells. The labeling density in the relay cell plasma membrane was about 3-fold higher than that of the interneurons. These results provide ultrastructural evidence for cell-type-specific expression levels and for uniform expression density of the α1G subunit over the plasma membrane of dLGN cells. J. Comp. Neurol. 518:4362–4374, 2010. © 2010 Wiley-Liss, Inc.

Perisomatic GABAergic input appears spared or even increased in intractable temporal lobe epilepsy, and has been suggested to contribute to the generation of pathological discharges. Nevertheless, its degree of functional activity during epileptiform synchronization has not been thoroughly investigated. Thus, it remains unclear how structural preservation or loss of domain-specific GABAergic input may affect the network. Here, we have taken advantage of a model of epileptiform activity in vitro to quantify the charge transfer provided by perisomatic GABAA receptor-mediated input to CA1 pyramidal neurons during interictal-like bursts. By recording both firing in GABAergic interneurons and the charge transfer generated by unitary postsynaptic currents to target pyramidal cells, we have estimated the charge transfer that would be dynamically generated by the recruitment of the entire pool of perisomatic-targeting interneurons and the number of perisomatic-targeting interneurons that would be required to generate the experimentally observed GABAergic input. In addition, we have recorded and compared the dynamics and charge density of GABAergic input recorded at different membrane compartments such as the soma vs. the proximal dendrite. Our results suggest that GABAA receptor-mediated perisomatic input is massively activated during burst synchronization and that its kinetic properties and charge density are similar at the soma and proximal dendrite. These functional results match structural data published by other laboratories very well and strengthen the hypothesis that the potential preservation of perisomatic GABAergic input in intractable epilepsies may be a key factor in the generation of pathological network activity.

In the mammalian retina, AII amacrine cells play a crucial role in scotopic vision. They transfer rod signals from rod bipolar cells to the cone circuit, and divide these signals into the ON and OFF pathways at the discrete synaptic layers. AII amacrine cells have been reported to generate tetrodotoxin (TTX)-sensitive repetitive spikes of small amplitude. To investigate the properties of the spikes, we performed whole-cell patch-clamping of AII amacrine cells in mouse retinal slices. The spike frequency increased in proportion to the concentration of glutamate puffer-applied to the arboreal dendrite and to the intensity of the depolarizing current injection. The spike activity was suppressed by L-2-amino-4-phosphonobutyric acid, a glutamate analogue that hyperpolarizes rod bipolar cells, puffer-applied to the outer plexiform layer. Therefore, it is most likely that the spike frequency generated by AII amacrine cells is dependent on the excitatory glutamatergic input from rod bipolar cells. Gap junction blockers reduced the range of intensity of input with which spike frequency varies. Application of TTX to the soma and the proximal dendrite of AII amacrine cells blocked the voltage-gated Na(+) current significantly more than application to the arboreal dendrite, indicating that the Na(+) channels are mainly localized in these regions. Our results suggest that the intensity of the glutamatergic input from rod bipolar cells is coded by the spike frequency at the soma and the proximal dendrite of AII amacrine cells, raising the possibility that the spikes could contribute to the OFF pathway to enhance release of neurotransmitter.

The onset of puberty is initiated by an increase in the release of the gonadotropin-releasing hormone (GnRH) from GnRH neurons in the hypothalamus. However, the precise mechanism that leads to the activation of GnRH neurons at puberty remains controversial. Spines are small protrusions on the surface of dendrites that normally receive excitatory inputs. In this study, we analyzed the number and morphology of spines on GnRH neurons to investigate changes in synaptic inputs across puberty in rats. For morphological estimation, we measured the diameter of the head (DH) of each spine and classified them into small-type (DH 0.65 μm), large-type (DH 0.65 μm) and giant-type (DH 0.9 μm). The greatest number of spines was observed at the proximal dendrite within 50 μm of the soma. At the soma and proximal dendrite, the number of spines was greater in adults than in juveniles in both male and female individuals. Classification of spines revealed that the increase in spine number was due to increases in large- and giant-type spines. To further explore the relationship between spines on GnRH neurons and pubertal development, we next analyzed adult rats neonatally exposed to estradiol benzoate, in which puberty onset and reproductive functions are disrupted. We found a decrease in the number of all types of spines. These results suggest that GnRH neurons become to receive more and greater excitatory inputs on the soma and proximal dendrites as a result of the changes that occur at puberty and that alteration to spines plays a pivotal role in normal pubertal development.

Recent theories have proposed that the unifying principle of brain function is the minimisation of variational free energy and that this is best achieved using a hierarchical predictive coding (HPC) framework. Hierarchical Temporal Memory (HTM) is a model of neocortical function that fits within the free energy framework but does not implement predictive coding. Recent work has attempted to integrate predictive coding and hierarchical message passing into the existing suite of HTM Cortical Learning Algorithms (CLA) producing a PC-CLA hybrid. In this paper we examine for the first time how such hierarchical message passing can be implemented in a pure HTM framework using distal dendrite structures that are already implemented in the CLA temporal pooler. We show this approach outperforms the more simplistic proximal dendrite structures used in the PC-CLA hybrid and also that the new CLA hierarchy is effective for anomaly detection and image reconstruction problems that are beyond the reach of the existing single-level CLA framework.

The mechanisms in the retina that generate light responses selective for the direction of image motion remain unresolved. Recent evidence indicates that directionally selective light responses occur first in the retina in the dendrites of an interneuron, i.e., the starburst amacrine cell, and that these responses are highly sensitive to the activity of Na-K-2Cl (NKCC) and K-Cl (KCC), two types of chloride cotransporter that determine whether the neurotransmitter GABA depolarizes or hyperpolarizes neurons, respectively. We show here that selective blockade of the NKCC2 and KCC2 cotransporters located on starburst dendrites consistently hyperpolarized and depolarized the starburst cells, respectively, and greatly reduced or eliminated their directionally selective light responses. By mapping NKCC2 and KCC2 antibody staining on these dendrites, we further show that NKCC2 and KCC2 are preferentially located in the proximal and distal dendritic compartments, respectively. Finally, measurements of the GABA reversal potential in different starburst dendritic compartments indicate that the GABA reversal potential at the distal dendrite is more hyperpolarized than at the proximal dendrite due to KCC2 activity. These results thus demonstrate that the differential distribution of NKCC2 on the proximal dendrites and KCC2 on the distal dendrites of starburst cells results in a GABA-evoked depolarization and hyperpolarization at the NKCC2 and KCC2 compartments, respectively, and underlies the directionally selective light responses of the dendrites. The functional compartmentalization of interneuron dendrites may be an important means by which the nervous system encodes complex information at the subcellular level.

The firing evoked by injection of simulated barrages of EPSCs into the proximal dendrite of layer 5 pyramidal neurons is greater than when comparable inputs are injected into the soma. This boosting is mediated by dendritic Na+conductances. However, the presence of other active conductances in the dendrites, some of which are nonuniformly distributed, suggests that the degree of boosting may differ along the somatodendritic axis. Here, we injected EPSC barrages at the soma and at the proximal, middle, and distal segments of the apical dendrite and measured boosting of subthreshold and suprathreshold responses. We found that although boosting was maintained throughout the apical dendrite, the degree of boosting changed nonmonotonically with distance from the soma. Boosting dipped in the middle dendritic segments as a result of the deactivation of the hyperpolarization-activated cation current,Ih, but increased in the distal dendrites as a result of the activation of Ca2+conductances. In the distal dendrites, EPSC barrages evoked repetitive bursts of action potentials, and the bursting pattern changed systematically with the magnitude of the input barrages. The quantitative changes in boosting along the somatodendritic axis suggest that inputs from different classes of presynaptic cells are weighted differently, depending on the location of the synaptic contacts. Moreover, the tight coupling between burst characteristics and stimulus parameters indicate that the distal dendrites can support a coding scheme that is different from that at sites closer to the soma, consistent with the notion of a separate dendritic integration site.

Maturation of specific neuronal connections in the mature nervous system includes elimination of redundant synapses formed earlier during development. In the cerebellum of adult animals, each Purkinje cell (PC) is innervated by a single climbing fiber (CF). In early postnatal development each PC is innervated by multiple CFs and elimination of synapses formed by supernumerary CFs occurs until monoinnervation is established at around postnatal day 20 (P20) in mice. It is not clear whether multiple CFs, or only a single CF, translocate from the cell body of immature PCs to the developing dendrite and, in case several CFs translocate, whether they share or segregate their innervation fields. To localize CF innervation fields, we imaged changes in postsynaptic sodium concentration resulting from CF-mediated postsynaptic currents. We found that more than one CF translocates from an innervation field on the cell body of the PC to the developing dendrite and that these CFs share rather than segregate their innervation fields. We concluded that both the soma and the proximal dendrite of the PC are territories of competition for the developing CFs and that the overlapping of their termination fields may be the prerequisite for a local process of elimination of all but one CF, as previously demonstrated in the developing neuromuscular junction.

Nogo-A is known to be a myelin-associated protein with strong inhibitory effect on neurite outgrowth and has been considered one of the major factors that hinder fiber regeneration in the central nervous system. Recent studies have demonstrated widespread occurrence of nogo-A mRNA and Nogo-A protein in neurons. Our concurrent immunohistochemical study substantiated the widespread distribution of neuronal Nogo-A. The present study was thus focused on its intraneuronal distribution in the central nervous system, using Western blotting, immunohistochemical, and immunogold electron microscopic techniques. Western blotting of the nucleus, cytoplasm, and membrane subcellular fractions of the cerebellum and spinal cord tissues demonstrated that all three fractions contained Nogo-A. Nogo-A immunoreactivity could be identified under confocal microscope in the nucleus, perikayon, and proximal dendrite and along the cell membrane. Under the electron microscope, the perikaryonal Nogo-A immunogold particles were mainly distributed at polyribosomes and rough endoplasmic reticulum, suggesting its relationship with translation process. The immunogold particles could also be found beneath or on the plasma membrane. In the nucleus, the Nogo-A immunogold particles were found to be localized at the chromatins of the nucleus, indicating its possible involvement in gene transcription. The presence of Nogo-A in the nucleus was further supported by transfection of COS-7L cells with nogo-A. This study provides the first immunocytochemical evidence for intraneuronal distribution of Nogo-A. Apparently, the significance of Nogo-A in the central nervous system is far more complex than what has been envisioned.

Cisternal stacks are induced during hypoxia, which may be associated with intracellular Ca2+ regulation. Although neurons are divided internally in different compartments, little is known about regional differences in cisternal stack formation. We investigated the effects of hypoxic hypoxia and later reoxygenation on cisternal stack formation and other ultrastructual changes in the proximal dendrite, dendritic spine, and cell body of cerebellar Purkinje cells in rats. After brief hypoxic events, cisternal stacks appeared predominantly in the proximal dendrites and after longer hypoxic events in dendritic spines and cell body. Following reoxygenation, cisternal stacks disappeared first in the cell body, followed by the dendritic spines, then the proximal dendrites. These results showed that stack formation occurred at different degrees and time courses among the three regions, and the effect was reversible, which suggests that these compartments are differentially sensitive to hypoxia.

Cortical columns contain specific neuronal populations with characteristic sets of connections. This wiring forms the structural basis of dynamic information processing. However, at the single-cell level little is known about specific connectivity patterns. We performed experiments in infragranular layers (V and VI) of rat somatosensory cortex, to clarify further the input patterns of inhibitory interneurons immunoreactive (ir) for vasoactive intestinal polypeptide (VIP). Neurons in acute slices were electrophysiologically characterized using whole-cell recordings and filled with biocytin. This allowed us to determine their firing pattern as regular-spiking, intrinsically bursting and fast-spiking, respectively. Biocytin was revealed histochemically and VIP immunohistochemically. Sections were examined for contacts between the axons of the filled neurons and the VIP-ir targets. Twenty pyramidal cells and five nonpyramidal (inter)neurons were recovered and sufficiently stained for further analysis. Regular-spiking pyramidal cells displayed no axonal boutons in contact with VIP-ir targets. In contrast, intrinsically bursting layer V pyramidal cells showed four putative single contacts with a proximal dendrite of VIP neurons. Fast-spiking interneurons formed contacts with two to six VIP neurons, preferentially at their somata. Single as well as multiple contacts on individual target cells were found. Electron microscopic examinations showed that light-microscopically determined contacts represent sites of synaptic interactions. Our results suggest that, within infragranular local cortical circuits, (i) fast-spiking interneurons are more likely to influence VIP cells than are pyramidal cells and (ii) pyramidal cell input probably needs to be highly convergent to fire VIP target cells.

The persistent Na(+) current (I(Nap)) is believed to be an important target of dopamine modulation in prefrontal cortex (PFC) neurons. While past studies have tested the effects of dopamine on I(Nap), the results have been contradictory largely because of difficulties in measuring I(Nap) using somatic whole-cell recordings. To circumvent these confounds we used the cell-attached patch-clamp technique to record single Na(+) channels from the soma, proximal dendrite (PD) or proximal axon (PA) of intact prefrontal layer V pyramidal neurons. Under baseline conditions, numerous well resolved Na(+) channel openings were recorded that exhibited an extrapolated reversal potential of 73 mV, a slope conductance of 14-19 pS and were blocked by tetrodotoxin (TTX). While similar in most respects, the propensity to exhibit prolonged bursts lasting40 ms was many fold greater in the axon than the soma or dendrite. Bath application of the D1/D5 receptor agonist SKF81297 shifted the ensemble current activation curve leftward and increased the number of late events recorded from the PD but not the soma or PA. However, the greatest effect was on prolonged bursting where the D1/D5 receptor agonist increased their occurrence 3 fold in the PD and nearly 7 fold in the soma, but not at all in the PA. As a result, D1/D5 receptor activation equalized the probability of prolonged burst occurrence across the proximal axosomatodendritic region. Therefore, D1/D5 receptor modulation appears to be targeted mainly to Na(+) channels in the PD/soma and not the PA. By circumventing the pitfalls of previous attempts to study the D1/D5 receptor modulation of I(Nap), we demonstrate conclusively that D1/D5 receptor activation can increase the I(Nap) generated proximally, however questions still remain as to how D1/D5 receptor modulates Na(+) currents in the more distal initial segment where most of the I Nap is normally generated.

The amplitude of backpropagating action potentials (BAPs) is attenuated, either activity- or neurotransmitter-dependently in the apical dendrite of hippocampal pyramidal neurons. To test the possibility that this BAP attenuation may contribute to regulating the inducibility of long-term potentiation (LTP), BAPs evoked by theta-burst stimulation (TBS), a standard protocol for LTP induction, to apical dendrite synapses were subjected to perturbation by conditioning stimuli to basal dendrite synapses. During this conditioned TBS (cTBS), the amplitude of BAPs was noticeably attenuated, but that of somatic action potentials was not. In the distal dendrite area, cTBS-induced LTP was much smaller than that induced by TBS. By contrast, no difference was observed between TBS- and cTBS-induced LTP in the proximal dendrite area. These findings suggest that the activity-dependent attenuation of BAPs, propagating along the apical dendrite, may serve to regulate hippocampal synaptic plasticity.

We investigated activity-dependent calcium increases in proximal dendrites of dentate granule cells in the rat hippocampus, and its relationship with induction of LTP at perforant path synapses (PP-synapses). LTP was induced at PP-synapses by high-frequency stimulation (HFS; 100 Hz for 0.4 s), and the same HFS evoked a dendritic calcium increase in the proximal dendrite. However, bath-application of the L-type voltage-dependent calcium channel (VDCC) blocker nimodipine noticeably reduced this calcium increase without abolishing induction of LTP. This calcium increase mediated by high-threshold VDCCs is likely to be evoked by action potentials. LTP induction at PP-synapses is hence suggested to be independent of action potential-induced calcium increases in the proximal dendrite.

From 1939 to 1967, J.L. Conel quantitatively studied the microscopic features of the developing human cerebral cortex and published the findings in eight volumes. We have constructed a database using his neuroanatomical measurements (neuronal packing density, myelinated large fiber density, large proximal dendrite density, somal breadth and height, and total cortical and cortical layer thickness) at the eight age periods (0 [term birth], 1, 3, 6, 15, 24, 48, and 72 postnatal months) he studied. In this report, we examine changes in neuron numbers over the eight age-points for 35 von Economo areas for which Conel gave appropriate data. From birth to 3 months postnatal age, total cortical neuron number increases 23–30%, then falls to within 3.5% of the birth value at 24 months, supporting our previous work showing that the observed decrease in the number of neurons per column of cortex under a 1-mm2 cortical surface from birth to 15 months is almost entirely due to cortical surface expansion. The present study also shows a 60–78% increase in total cortical neuron number above the birth value from postnatal ages 24 to 72 months. The generalization, to humans at least, of the finding of no postnatal neurogenesis in rhesus macaques, a species belonging to a super-family that diverged from that of Homo sapiens more than 25 million years ago, is not warranted until explicitly proven for humans. The data of the present study support the existence of substantial postnatal neurogenesis in humans for the 35 cortical areas studied.

The distribution of glutamate receptor subtypes on the surface of neurons is highly relevant for synaptic activation and signal processing in the neocortex. As a novel approach we have used infra-red videomicroscopy in combination with photostimulation or microiontophoresis in brain slices of rat neocortex to map the distribution of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors on pyramidal neurons of layer V. Both modes of application revealed a spatially distinct distribution of glutamate receptor subtypes: the soma and the proximal dendrite of neurons are highly sensitive to NMDA, whereas the more distal parts of the dendrite are more sensitive to AMPA. An implication is that NMDA receptors near the soma might regulate the amplification of synaptic signals resulting from AMPA receptor activation on remote dendritic sites.

A multi-compartmental molecular model has been developed for rodent basal forebrain cholinergic neurons with established gene expression levels. Reconstruction of neurons and network function were acquired using the Transcriptome-To-Physiome™ (TTP™) NeurobioSimulation. Gene expression values [NCBI GEO GSE 13379] were used to derive protein level and kinetic parameters for ligand and voltage gated ion channels in the TTP™ NeurobioSimulator Model using COPASI® software. Global parameters for membrane potential used permeability and ion concentrations inside and outside of the membrane in the Goldman-Hodgkin-Katz equation. Four compartments of the model neuron are included: glutamate synapse, distal dendrite, proximal dendrite, and axon hillock. The simulation of a voltage-gated sodium channel activation, and inactivated states of distal dendrites of cholinergic modeled neurons depends on the excitatory postsynaptic potential (EPSP) event. This distally activated event yielded temporally relevant proximal dendritic activation and inactivation of voltage-gated sodium and potassium channels in the reconstructed neuron. Graded potentials showed temporal summation and a classic action potential occurs at the axon hillock with sodium and potassium fluxes as expected. In future studies, we will reconstruct the electrophysiology of vulnerable neuronal populations in the diseased brain and compare them to controls thus lending substantial insight into molecular and network function corollary to neuropathogenesis.

The coding of odor intensity by an olfactory receptor neuron model was studied under steady-state stimulation. Our model neuron is an elongated cylinder consisting of the following three components: a sensory dendritic region bearing odorant receptors, a passive region consisting of proximal dendrite and cell body, and an axon. First, analytical solutions are given for the three main physiological responses: (1) odorant-dependent conductance change at the sensory dendrite based on the Michaelis-Menten model, (2) generation and spreading of the receptor potential based on a new solution of the cable equation, and (3) firing frequency based on a Lapicque model. Second, the magnitudes of these responses are analyzed as a function of odorant concentration. Their dependence on chemical, electrical, and geometrical parameters is examined. The only evident gain in magnitude results from the activation-to-conductance conversion. An optimal encoder neuron is presented that suggests that increasing the length of the sensory dendrite beyond about 0.3 space constant does not increase the magnitude of the receptor potential. Third, the sensitivities of the responses are examined as functions of (1) the concentration at half-maximum response, (2) the lower and upper concentrations actually discriminated, and (3) the width of the dynamic range. The overall gain in sensitivity results entirely from the conductance-to-voltage conversion. The maximum conductance at the sensory dendrite appears to be the main tuning constant of the neuron because it determines the shift toward low concentrations and the increase in dynamic range. The dynamic range of the model cannot exceed 5.7 log units, for a sensitivity increase at low odor concentration is compensated by a sensitivity decrease at high odor concentration.

Although the majority of mRNAs expressed in neurons are confined to the perikaryon, a growing number appear to be transported into dendrites. It is likely that this allows for the local regulation of protein synthesis within discrete subcellular compartments. Here, three different subcellular distribution patterns are demonstrated for four mRNAs that encode proteins highly expressed in Purkinje cells and their dendrites; mRNAs are found in the perikaryon only, perikaryon and proximal dendrite, or perikaryon and proximal plus distal dendrites. Further, it is shown that transport of an mRNA into the dendrites increases its sensitivity to translational inhibition by diphtheria toxin. These data suggest a simple model whereby the transport machinery can regulate the translation of selected mRNAs. Thus, environmental signals that generally affect translational efficiency in concert with the selectivity provided by the transport machinery could provide a means to locally regulate the synthesis of a restricted pool of proteins.

To examine synaptic input and association of terminals containing dopamine and other transmitters to rat striatal nitric oxide synthase-expressing neurons, an electron microscopic study using tyrosine hydroxylase (TH) immunohistochemistry combined with histochemistry for NADPH-diaphorase (NADPHd) was performed. NADPHd-positive neurons had medium-sized cell bodies containing a highly invaginated nucleus and received relatively sparse synaptic input; 3.6% of boutons apposed to the NADPHd-positive neurons were TH-immunoreactive. Of these TH-immunoreactive boutons, two synaptic contacts showing symmetrical synaptic specializations were found on a cell body and a proximal dendrite of a NADPHd-positive neuron. Other nonsynaptic TH-immunoreactive boutons were occasionally associated with unlabeled terminals adjacent to the NADPHd-positive dendrites and also forming asymmetric synaptic contacts with unlabeled spinous or dendritic profiles. These results suggest that activity of the striatal neurons that release nitric oxide may be regulated by direct synaptic input from dopaminergic neurons and also suggest that the TH-immunoreactive terminals associated with the dendrites of nitric oxide synthase-expressing neurons provide the sites where nitric oxide influences dopamine release from neighboring terminals.

The calcium-binding protein calretinin is present in an intrinsic GABAergic and an extrinsic non-GABAergic system in the rat and monkey hippocampal formation. Important species differences have been noted in hippocampal cell types immunostained for calretinin and the termination pattern of calretinin containing hypothalamic afferents in the hippocampus. In the present study, calretinin-containing neurons were visualized using immunocytochemistry in the human hippocampal formation of individuals which showed no significant neuropathological alterations. Calretinin-immunoreactivity was present exclusively in non-granule cells of the dentate gyrus and in non-pyramidal cells of Ammon's horn. Calretinin-positive neurons were found most frequently in the hilus of the fascia dentata and in strata radiatum and lacunosum-moleculare of CA1, whereas neurons in CA2 and CA3 were rarely immunostained. The majority of calretinin-immunoreactive neurons were small, bipolar or fusiform neurons. The dendritic trees of the calretinin-positive neurons were, for the most part, parallel to the dendrites of the principal cells. In the hilus, however, we observed cells with dendrites restricted to the hilar area. These dendrites were parallel to the granule cell layer. In the stratum lacunosum-moleculare, neurons with dendrites oriented parallel to the hippocampal fissure were frequently detected. In general, dendrites were smooth or sparsely spiny, displaying small conventional spines. The axons usually emerged from the proximal dendrite and could be followed over long distances. Axons were thin, had small varicosities and displayed only few collaterals which branched relatively far away from the cell body. Distinct bands of darkly stained calretinin-positive fibers occupied the innermost portion of the dentate molecular layer and the pyramidal cell layer of CA2. This distribution of calretinin-immunoreactive structures in the human hippocampus is similar to that observed in other primates but differs from that described in lower mammals, i.e., the rat. Our findings suggest that primates may share a common hippocampal calretinin-containing system, presumably both the intrinsic GABAergic and the extrinsic hypothalamic non-GABAergic components.

In this quantitative electron microscopic study we investigated the distribution of GABA axon terminals on rat abducens motoneurons by combining retrograde labelling of montoneurons with post-embedding immunodetection of GABA. We analysed the synapses on 13 cell bodies and 60 proximal dendritic profiles distributed along the entire rostro-caudal extent of the nucleus. For each of these two compartments, we analysed 1754 and 1176 axon terminals in contact with 6042 and 3299 microns of postsynaptic membrane. The axon terminals were classified as Sv-type (containing spherical vesicles) or Pv-type (containing pleomorphic vesicles). The GABAergic terminals contained pleomorphic vesicles and established mainly symmetrical synaptic contacts. Their apposition lengths were greater than those of unlabelled terminals. On cell bodies, the percentage of GABAergic synaptic covering varied from 2.5% to 14.1% and the synaptic frequency of GABAergic axon terminals varied from 0.6% to 8.9%. These two parameters were significantly correlated with the diameter of the motoneurons. The percentage of synaptic covering and synaptic frequency were smaller on dendrites of small motoneurons than on those of large ones. The proximal dendrites of small motoneurons had a lesser GABAergic innervation than large ones. The total synaptic covering and frequency were smaller on somata than on dendrites. However, the percentage of synaptic covering by GABA terminals was higher on cell bodies than on proximal dendrites.

Previous patch-clamp studies by our laboratory showed that acute exposure to the pesticide rotenone augments inward currents evoked by N-methyl-d-aspartate (NMDA) in substantia nigra zona compacta (SNC) dopamine neurons in slices of rat brain. The present experiments were done to search for histological evidence of increased neurotoxicity produced by combined rotenone and NMDA treatments. In horizontal slices of rat midbrain, we found that a 30 min superfusion with 100 nM rotenone caused significant injury to tyrosine hydroxylase (TH)-positive proximal dendrites in dorsal and ventral regions of the SNC and ventral tegmental area (VTA). Moreover, treatment with 100 μM NMDA potentiated rotenone toxicity. In contrast, treatment with 30 μM NMDA protected against rotenone-induced injury to dendrites in the ventral SNC and ventral VTA. Interestingly, treatment with 30 μM NMDA-alone produced an apparent increase in proximal dendrite scores in ventral SNC and dorsal VTA. We conclude that NMDA has concentration-dependent actions on rotenone toxicity that differ according to regional subtype of dopamine neuron.

The synaptic input of six spiny stellate neurons in sublamina 4A of cat area 17 was assessed by electron microscopy. The neurons were physiologically characterized and filled with horseradish peroxidase in vivo. After processing the neurons were reconstructed at the light microscopic level using computer-assisted methods and analyzed quantitatively. The extensive branching of the dendritic tree about 50 microns from the soma meant that the distal branches constituted five times the length of proximal dendrite. Proximal and distal portions of a single dendrite from each neuron were examined in series of ultrathin sections (1,456 sections) in the electron microscope. The majority (79%) of the 263 synapses examined were asymmetric; the remainder (21%) were symmetric. Symmetric synapses formed 35% of synapses sampled on proximal dendrites and were usually located on the shaft. They formed only 4% of synapses sampled on distal dendrites. Spines accounted for less than half of the total asymmetric synapses (45%); the remainder were on shafts. Symmetric synapses formed with four of 92 spines. Nine spines formed no synapses. Spiny stellate neurons in cat visual cortex appear to differ considerably from pyramidal neurons in having a significant asymmetric (excitatory) synaptic input to the dendritic shaft.

SummaryCortical pyramidal neurons receive thousands of synaptic inputs arriving at different dendritic locations with varying degrees of temporal synchrony. It is not known if different locations along single cortical dendrites integrate excitatory inputs in different ways. Here we have used two-photon glutamate uncaging and compartmental modeling to reveal a gradient of nonlinear synaptic integration in basal and apical oblique dendrites of cortical pyramidal neurons. Excitatory inputs to the proximal dendrite sum linearly and require precise temporal coincidence for effective summation, whereas distal inputs are amplified with high gain and integrated over broader time windows. This allows distal inputs to overcome their electrotonic disadvantage, and become surprisingly more effective than proximal inputs at influencing action potential output. Thus, single dendritic branches can already exhibit nonuniform synaptic integration, with the computational strategy shifting from temporal coding to rate coding along the dendrite.

1. Flash photolysis of caged cyclic nucleotides was used to examine the contribution of the ciliary cyclic nucleotide-gated conductance to olfactory transduction in the tiger salamander. Brief illumination of solitary olfactory receptor cells loaded with 100 microM caged cyclic AMP caused a large inward current (peak amplitude 355 +/- 200 pA; mean +/- S.D. for eleven cells) under whole-cell voltage clamp at -50 mV. 2. The photolysis response was initiated after a latency of 4-12 ms, whereas an odorant response of identical amplitude had a latency of several hundred milliseconds. The amplitudes of both responses exhibited almost identical voltage dependence between -50 and +25 mV, with both reversing near 0 mV. The time courses of the falling phases of odorant and photolysis responses also exhibited similar voltage dependence, both being prolonged at positive voltages. 3. Photolysis of caged cyclic GMP activated a current similar in amplitude and time course to that produced by photolysis of caged cyclic AMP. 4. When the flash was spatially limited to the cilia, the amplitude and duration of the photolysis response increased linearly with the length of the cilia illuminated (for cilia not longer than 30-40 microns) while the latency remained constant at 4-12 ms. The increase in duration was described semi-quantitatively by a model which incorporated diffusion and saturable hydrolysis of cyclic AMP. When the flash was limited to the soma or proximal dendrite, the response latency was proportional to the square of the distance between the illuminated region and the cilia. 5. Dialysis of cells with 500 microM cyclic AMP from a whole-cell electrode under voltage clamp activated a large transient inward current. Simultaneous suction electrode recording showed that this current originated almost entirely from the ciliary membrane. The density of cyclic nucleotide-gated channels was estimated to be 800-fold higher in the cilia than in the soma. 6. Summation of simultaneous odorant and photolysis responses was non-linear, the flash-induced current being enhanced during a small odorant response and attenuated during a large odorant response. Summation of two photolysis responses was similarly non-linear. The data were consistent with odorant stimuli and cyclic AMP both activating a common cyclic nucleotide-gated conductance with a Hill coefficient, n, of 2.0-4.4. For n = 2.5, the basal cyclic AMP concentration was estimated to be less than 20% of the K 1/2, which predicts a basal current of 5.8 pA, less than 2% of the maximum.(ABSTRACT TRUNCATED AT 400 WORDS)

DEG/ENaC channels have been broadly implicated in mechanosensory transduction, yet many questions remain about how these proteins contribute to complexes that sense mechanical stimuli. In C. elegans, two DEG/ENaC channel subunits are thought to contribute to a gentle touch transduction complex: MEC-4, which is essential for gentle touch sensation, and MEC-10, whose importance is less well defined. By characterizing a mec-10 deletion mutant, we have found that MEC-10 is important, but not essential, for gentle touch responses in the body touch neurons ALM, PLM, and PVM. Surprisingly, the requirement for MEC-10 in ALM and PLM is spatially asymmetric; mec-10 animals show significant behavioral and physiological responses to stimulation at the distal end of touch neuron dendrites, but respond poorly to stimuli applied near the neuronal cell body. The subcellular distribution of a rescuing MEC-10::GFP translational fusion was found to be restricted to the neuronal cell body and proximal dendrite, consistent with the hypothesis that MEC-10 protein is asymmetrically distributed within the touch neuron process. These results suggest that MEC-10 may contribute to only a subset of gentle touch mechanosensory complexes found preferentially at the proximal dendrite.

Calcium is known to regulate several phenomena like neuronal excitability and plasticity. Interestingly, the spatiotemporal profile of dendritic calcium depends on several processes, specific to each neuronal type. In this study, we investigated Ca(2+) buffering and action potential (AP)-evoked Ca(2+) signaling in the dendrites of anatomically identified oriens lacunosum-moleculare (O-LM) cells, a major type of dendrite-targeting interneurons in the hippocampal CA1 region, using a combination of whole-cell patch-clamp recording and fast Ca(2+) imaging in acute rat brain slices. Cells were loaded with fluorescent Ca(2+) indicators fura-2 or Oregon Green BAPTA-1 (OGB-1) via patch-clamping electrode, and the effect of fura-2 on AP-evoked dendritic Ca(2+) transients was determined by ratiometric Ca(2+) imaging. To estimate intracellular Ca(2+) concentrations ([Ca(2+)](i)) and endogenous Ca(2+)-binding ratio (kappa(s)) in the proximal dendrite, fluorescence signals were converted into [Ca(2+)](i) using the ratioing method and were analyzed on the basis of the "single compartment model." Resting [Ca(2+)](i) was 22+/-5 nM and the build-up of [Ca(2+)](i) during a single AP was up to 656+/-226 nM. Analysis of Ca(2+) transients revealed that O-LM cells have a relatively low endogenous Ca(2+)-binding ratio (kappa(s)): the kappa(s) was 20+/-8 estimated during fura-2 loading and 27 estimated under steady-state fura-2 concentrations, respectively. To further examine the spatial profile of dendritic Ca(2+) transients, we measured somatic AP-evoked Ca(2+) transients beyond proximal dendrites using OGB-1. Dendritic Ca(2+) transients evoked by single APs or AP trains are not limited to regions close to the soma. The amplitude and decay of [Ca(2+)](i) associated with backpropagating APs are relatively independent of the distance from the soma. In sum, O-LM cells exhibit low endogenous Ca(2+)-binding ratios and relatively distance-independent Ca(2+) dynamics in the dendrites. These special features of Ca(2+) signaling in O-LM cells may have important functional implications for both normal and pathological conditions.

We investigated competition between synapses located proximal and distal dendrites through STDP learning rules using a dentate granule cell model. The proximal dendrite and the distal dendrite were stimulated by a regular pulse train and a random pulse train respectively. When both synapses were subject to asymmetric STDP rules, the distal synapse was not enhanced but the proximal synapse was enhanced through synaptic competition. This competition was caused when the integral of the LTD window was larger than that of the LTP window of the STDP rule in the distal dendrite. In contrast, when the proximal synapse was not subject to the asymmetric STDP rule but a Mexican-hat STDP rule, location of the synapse enhanced by synaptic competition was switched from the proximal synapse to the distal synapse. We also examined the role of inhibitory interneurons in the synaptic competition.

The presence of tetrodotoxin (TTX)-sensitive slowly-inactivating Na+ channels in the dendrites of neocortical layer 5 neurons was tested by focal iontophoresis of glutamate on the dendrite while voltage clamping the soma and proximal dendrite. The glutamate-transmitted current was measured with the voltage clamp circuit. When the soma was depolarized the transmitted current increased indicating voltage-dependent properties in the dendrite. Over 50% of this increased voltage-dependence was blocked by TTX indicating a large portion of the enhanced dendro-somatic current was caused non-inactivating Na+ channel inward rectification. The glutamate-transmitted current measured with a voltage clamp of the soma at firing level was equal to the effective glutamate measured during repetitive firing.

In spinal neurons, plateau potentials serve to amplify neuronal input signals. To a large extent, the underlying persistent inward current is mediated by a subtype of the L-type calcium channel (Ca(V)1.3). In the present investigation, we have studied its distribution and cellular localization in the cat spinal cord by light and electron microscopic immunohistochemistry. The results show that Ca(V)1.3-like immunoreactivity is widely distributed in all segments of the spinal cord but that the distribution in the different laminae of the spinal gray matter varies, with the highest density of labeled neurons in lamina IX and the lowest in lamina II. The labeling intensity was highest in neuronal somata, but a certain length of the proximal dendrite was also labeled. Some neuronal groups exhibited a particularly dense labeling; these include the lateral motoneuronal group in the cervical and the lumbar enlargements and the phrenic nucleus in cervical, Clarke's nucleus in lower thoracic and upper lumbar, and Onuf's nucleus in upper sacral segments. At the ultrastructural level, Ca(V)1.3-immunoreactive products were found in neuronal somata and dendrites of different sizes. In the soma, they were predominantly associated with the rough endoplasmic reticulum but some also with the plasma membrane. In dendrites, they were associated with both intracellular organelles, including microtubules and microchondria, and the plasma membrane. These results indicate that significant proportions of the neurons in cat spinal cord, including projection neurons, interneurons, and motoneurons, are endowed with ion channels that subserve persistent inward currents and act to amplify synaptic input signals.

The thalamic interneuron exemplified by that found in the cat’s lateral geniculate nucleus provides a potent GABAergic and thus inhibitory input to relay cells. It thus plays a key role in controlling the flow of information to cortex. The synaptic inputs from these interneurons to relay cells are particularly interesting for two reasons. First, the interneuron appears to employ two independent input/output routes: a conventional axonal one that integrates inputs onto the cell body and proximal dendrites; and an unconventional one involving dendritic outputs that are both presynaptic and postsynaptic. Cable modeling suggests that the dendritic output route is organized into numerous, functionally independent streams that are also independent of the cell body and thus action potential generation. Second, the dendritic outputs in addition to being presynaptic to relay cell dendrites, are postsynaptic chiefly to either retinal or parabrachial inputs. Details of these output synapses, which involve complex circuits known as triads found widely throughout thalamus, lead to rather speculative but testable ideas regarding how interneurons help modulate relay cell activity. It is hoped that following through some of these ideas will provide genuine insights into the functioning of this circuitry that appears key to thalamic relays.

In this study, to identify the precise localization of the muscarinic receptor subtypes m2, m3 and m4 in the rostral part of the rat reticular thalamic nucleus (rRt), namely, the limbic sector, we used receptor-subtype-specific antibodies and characterized the immunolabeled structures by light, confocal laser scanning, and electron microscopies. The m2-immunolabeling was preferentially distributed in the distal dendrite region where cholinergic afferent fibers tend to terminate and in the peripheral region of somata, whereas the m3-immunolabeling was more preferentially distributed in a large part of somata and in proximal dendrite shafts than in the distal dendrite region. Dual-immunofluorescence experiments demonstrated that majority of rRt neurons with parvalbumin immunoreactivity contain both m2 and m3. Neither m2 nor m3 was detected in presynaptic terminals or axonal elements. No m4-immunolabeling was detected in the rostral part of the thalamus including rRt. These results show the different distributions of m2 and m3 in rRt neurons, and strongly suggest that m2 is more closely associated with cholinergic afferents than m3.

We are investigating the computational properties of principal neurons in the mammalian brain. To manage the small size and intricate structure of neuronal dendrites, we employ advanced optical imaging techniques in combination with automatic image reconstruction and computational modeling to study their complex spatio-temporal pattern of activity. I. INTRODUCTION HE mammalian brain contains some 10 10 individual neurons that are highly inter-connected, each receiving input from somewhere between 10 2 and 10 5 other neurons. It is currently well-accepted that the information processing capability of the brain is not only based on the large number of neurons and their high connectivity, but also on the performance of individual neurons. Particularly, our view of the neuronal dendrite has changed from that of electric cables, passively conducting postsynaptic signals to the cell body, to that of active neuronal structures, capable of complex spatio-temporal electrochemical dynamics. Therefore, there exists a keen interest in understanding the information processing capability of a single neuron. The structural and functional analysis of individual neurons is complicated by their intricate shapes, small sizes, and fast response times. Traditionally, structure was observed and documented with histological techniques that require fixing, staining and physical micro-sectioning of brain tissue. Function was monitored with micro-pipettes; for technical reasons, their access was mainly limited to the cell body or the proximal dendrite. Today, sophisticated optical imaging techniques are increasingly employed for studying neurons, replacing these invasive approaches. The strong light scattering properties of living brain tissue

Spines can grow and retract within hours of activity perturbation. We investigated the time course of spine formation in a model of plasticity involving changes in brain architecture where spines of a dendritic domain become innervated by a different neuronal population. Following a lesion of rat olivocerebellar axons, by severing the inferior cerebellar peduncle, new spines grow on the deafferented proximal dendrite of the Purkinje cells (PCs) and these new spines become innervated by parallel fibres (PFs) that normally contact only the distal dendrites. The varicosities of climbing fibre (CF) terminal arbors disappear within 3 days of the lesion. Spine density in the proximal dendritic domain begins to rise within 3 days and continues to increase towards a plateau at 6-8 days. In 'slow Wallerian degeneration' mice, in which axonal degeneration is delayed, climbing fibre varicosities virtually disappear at 14 rather than 3 days. Spine density in the proximal dendritic domain is similar to control Purkinje cells up to 14 days and increases significantly 18 days postlesion. The delayed spinogenesis in the latter mutant is the result of a persistence of the climbing fibre presynaptic structure in the absence of activity. Therefore, climbing fibre activity itself is not directly responsible for the suppression of spine formation, but suppression mechanisms tend to become weaker as long as the structural dismantling of the presynaptic varicosities proceeds. Thus, spinogenesis is guided by two different mechanisms; a rapid one related to changes in homotypic remodeling and a slower one, which requires the removal of a competitive afferent.

By blocking electrical activity in the cerebellar cortex the Purkinje cell dendrites become a uniform territory with a high density of spines all bearing the glutamate receptor δ2 subunit (GluRδ2) and being mainly innervated by parallel fibers. Such a subunit, which is constitutively targeted specifically to the parallel fiber synapses, appears in the spines contacted by the climbing fibers before they disconnect from the target. A similar pattern of hyperspiny transformation and innervation occurs a few days after a subtotal lesion of the inferior olive, the source of climbing fibers. During the climbing fiber reinnervation process which follows the removal of the electrical block or by collateral sprouting of surviving inferior olive neurons, the new active climbing fibers establish synaptic contacts with proximal dendritic spines that bear GluRδ2s. After, they repress these subunits and displace the parallel fibers to the distal dendritic territory. These findings suggest the following operational principle in the axonal competition for a common target. The Purkinje cells have an intrinsic phenotypic profile which is compatible with the parallel fiber innervation, this mode being operational in targets innervated by a single neuronal population, like the neuromuscular system. An additional input, the climbing fibers, in order to achieve its own territory on the proximal dendrite needs the ability to displace the competitor. Such an inhibition is activity-dependent and the activity needs to be present in order to allow the climbing fiber to maintain its territory, even when the developmental period is over.

Structure and function are inextricably wedded, a principle extending far beyond studies of nervous systems. Nowhere is this principle more profoundly and consistently exemplified than it is in nervous systems, however. It is clear, for example, from the diversity of cell types, the specializations of portions of cells, as well as the various contacts and intercellular juxtapositions, that functional diversity can be predicted from structural specialization. Thus, it is easy to understand that spinal motoneurons, with their long myelinated axons, carry out a different set of functions than do neurons that are devoid of axons, such as retinal amacrine cells. The former are engaged in high-speed transfer of information over long distances; the latter are not. Still, these two cell types remain neurons that integrate and process the information that they are specialized to receive and transmit. More subtle variations in neuron types than those gross differences seen between motoneurons and amacrine cells have been appreciated for some time now, of course. The presence or absence of spines on dendrites is an immediate clue to functional diversity among otherwise similar neurons, as is the presence or absence of microvesicles in dendrites, or whether the cell’s axon arises from the soma or from a proximal dendrite. This tight structure-function relationship holds not only for the individual cells themselves, but also for the larger structures formed by cell aggregates, e.g., the juxtapositions of cells within a nucleus in the brain. Such juxtapositions are known to be dynamic, even in the mature animal.

Objectives:To characterise the neuropathological phenotypes of two affected individuals from a family with an unusual clinical phenotype resembling motor neurone disease and dementia. Methods:Histological sections of cerebral cortex, basal ganglia, brain stem, cerebellum, and spinal cord were stained with haematoxylin-eosin, luxol fast blue, silver stains, anti-tau, anti-ubiquitin, anti-α-synuclein, and anti-neurofilament. Results:Numerous ubiquitin positive, tau and α-synuclein negative intraneuronal inclusions were present in the cerebral cortex (particularly within the dentate gyrus), cerebellar cortex, brain stem, and spinal cord. The cerebellar ubiquitinated inclusions were located in the proximal dendrite of the Purkinje cells. Loss of Purkinje cells and occasional silver and neurofilament positive axonal swellings (torpedoes) were also seen within the cerebellar cortex. The main difference between the two cases was the severity of the spinal cord involvement: no significant pathology was present within one, but obvious motor neurone disease within the other. Conclusions:The clinical and neuropathological findings in this family are best described as an example of familial motor neurone disease with dementia. Intraneuronal ubiquitin inclusions together with agyrophilic, neurofilament positive torpedoes were present within the cerebellar cortex, both previously unrecognised findings in this group of diseases.

Purkinje cells have two action potentials: Climbing fiber responses (CFRs) and simple spikes (SSs). CFRs reflect the discharge of a single climbing fiber at multiple synaptic sites on the proximal dendrite of the Purkinje cell. SSs reflect the summed action of a subset of parallel fiber synapses on Purkinje cell dendritic spines. Because mossy fiber afferents terminate on granule cells, the ascending axons of which bifurcate, giving rise to parallel fibers, the modulation of SSs has been attributed to mossy fiber afferent signals. This inference has never been tested. Conversely, the low discharge frequency of CFRs has led many to conclude that they have a unique and intermittent role in cerebellar signal processing. We examine the relative potency of vestibularly modulated mossy fiber and climbing fiber signals in evoking CFRs and SSs in Purkinje cells of the uvula-nodulus in chloralose-urethane-anesthetized rabbits. Vestibular primary afferents were blocked by unilateral labyrinthectomy (UL). A UL destroys the vestibular primary afferent signal to the ipsilateral uvula-nodulus, while leaving intact the vestibular climbing fiber signal from the contralateral inferior olive. After UL, vestibular stimulation modulated CFRs and SSs in ipsilateral uvula-nodular Purkinje cells, demonstrating that the primary vestibular afferent mossy fiber input to the ipsilateral uvula-nodulus was not necessary for SS modulation. Unilateral microlesions of the caudal half of the β-nucleus of the inferior olive reduced a modulated climbing fiber signal to the contralateral uvula-nodulus, causing loss of both vestibularly modulated CFRs and SSs in contralateral Purkinje cells. Vestibular climbing fibers not only evoke low-frequency CFRs, but also indirectly modulate higher-frequency SSs. This modulation must be attributed to cerebellar interneurons. Golgi cell inhibition of granule cells may provide the interneuronal mechanism for CFR-induced SS modulation.

We characterized the properties and functional roles of voltage-dependent potassium channels in the dendrites of Purkinje neurons studied in rat cerebellar slices. Using outside-out patches formed ≤250 μm away from the soma, we found that depolarization-activated potassium channels were present at high density throughout the dendritic tree. Currents required relatively large depolarizations for activation (midpoint, approximately –10 mV), had rapid activation and deactivation kinetics, and inactivated partially (20–70% over 200 msec) with both fast (time constant, 15–20 msec) and slow (300–400 msec) components. Inactivating and noninactivating components were both blocked potently by external tetraethylammonium (half-block by 150 μm) and 4-aminopyridine (half-block by 110 μm). The voltage dependence, kinetics, and pharmacology suggest a predominant contribution by Kv3 family subunits, and immunocytochemical experiments showed staining for both Kv3.3 and Kv3.4 subunits in the dendritic tree. In the proximal dendrite, potassium channels were activated by passively spread sodium spikes recorded at the same position, and experiments using dual recordings showed that the channels serve to actively dampen back-propagation of somatic sodium spikes. In more distal dendrites, potassium currents were activated by voltage waveforms taken from climbing fiber responses, suggesting that they help shape these responses as well. The requirement for large depolarizations allows dendritic Kv3 channels to shape large depolarizing events while not disrupting spatial and temporal summation of smaller excitatory postsynaptic potentials.

There is a marked increase in the maternal behavior displayed by a female rat following pregnancy-due primarily to exposure to the gonadal hormones progesterone and estradiol (P and E(2), respectively). We examined Golgi-Cox silver-stained, Vibratome-sectioned neurons visualized and traced using computerized microscopy and image analysis. In Part One, we examined the hormonal-neural concomitants in the medial preoptic area (mPOA), an area of the brain that regulates maternal behavior, by comparing cell body size (area in microm(2); also referred to as soma and perikaryon) in the mPOA and cortex of five groups (n = 4-6/group) of ovariectomized (OVX-minus), diestrous, sequential P and E(2)-treated (P+E(2)), late-pregnant, and lactating rats; for Part Two, we examined a subset of mPOA neurons, which were traced in their entirety, from these same subjects. In Part One, whereas there was no difference between OVX-minus and diestrous females, both had smaller somal areas compared to OVX+P+E(2)-treated and late-pregnant females. The area of the soma returned to diestrous/OVX-minus levels in the lactating females. We found no change among the five groups in area of cell body in cortical neurons, which generally lack steroid receptors. In Part Two, which included a more detailed morphometric analysis of mPOA neurons, we examined several additional measures of dendritic structure, including number of proximal dendritic branches (the largest proximal dendrite was defined as the one with the largest diameter leaving the soma); cumulative length of the largest proximal dendrite; area of the cell body; number of basal dendrites; cumulative basal dendritic length; number of basal dendritic branches; and branch-point (distance from cell body to first branch of largest proximal dendrite). Again, we found similar effects on cell body size as in Part One, together with effects on number of basal dendritic branches and cumulative basal dendritic length in pregnant and P+E(2)-treated groups compared to OVX, diestrous, and lactating. An increase in somal area denotes increased cellular activity, and stimulatory effects on additional neuronal variables represents modifications in information processing capacity. Pregnancy and its attendant hormonal exposure, therefore, may stimulate neurons in the mPOA, which then contribute (in an as yet undetermined manner) to the display of maternal behavior. During the postpartum lactational period, when cues from pups primarily maintain maternal attention, the neuronal soma appears to return to a pre-pregnancy, non-hormonally dependent state, whereas other aspects of the dendrite remain altered. Collectively, these data demonstrate a striking plasticity in the brains of females that may be reflected in modifications in behavior.