Axon Collaterals Research Articles

Cortical electrical stimulation in female rats with a cervical spinal cord injury to promote axonal outgrowth.

Electrical stimulation (ES) to promote corticospinal tract (CST) repair after spinal cord injury (SCI) is underinvestigated. This study is the first to detail intracortical ES of the injured CST. We hypothesize that cortical ES will promote CST collateralization and regeneration, prevent dieback, and improve recovery in an SCI rat model. The CST was transected at the the fourth cervical level in adult female Lewis rats trained in a stairwell grasping task. Animal groups included (a) ES333 (n = 14; 333 Hz, biphasic pulse for 0.2-ms duration every 500 ms, 30 pulses per train); (b) ES20 (n = 14; 20 Hz, biphasic pulse for 0.2-ms duration every 1 s, 60 pulses per train); (c) SCI only (n = 10); and (d) sham (n = 10). ES of the injured forelimb's motor cortex was performed for 30 min immediately prior to SCI. Comparisons between histological data were performed with a 1-way ANOVA or Kruskal-Wallis test, and grasping scores were compared using repeated-measures 2-way ANOVA. Significantly more axonal collateralization was found in ES333 animals compared with controls (p < .01). Axonal dieback analysis revealed ES20 rats to have consistently more dieback than the other groups at all points measured (p < .05). No difference in axonal regeneration was found between groups, nor was there any difference in functional recovery. Cortical ES of the injured CST results in increased collateral sprouting and influences neuroplasticity depending on the ES parameters used. Further investigation regarding optimal parameters and its functional effects is required.

Neural circuits underlying jaw movements for the prey-catching behavior in frog: distribution of vestibular afferent terminals on motoneurons supplying the jaw.

Coordinated movement of the jaw is essential for catching and swallowing the prey. The majority of the jaw muscles in frogs are supplied by the trigeminal motoneurons. We have previously described that the primary vestibular afferent fibers, conveying information about the movements of the head, established close appositions on the motoneurons of trigeminal nerve providing one of the morphological substrates of monosynaptic sensory modulation of prey-catching behavior in the frog. The aim of our study was to reveal the spatial distribution of vestibular close appositions on the somatodendritic compartments of the functionally different trigeminal motoneurons. In common water frogs, the vestibular and trigeminal nerves were simultaneously labeled with different fluorescent dyes and the possible direct contacts between vestibular afferents and trigeminal motoneurons were identified with the help of DSD2 attached to an Andor Zyla camera. In the rhombencephalon, an overlapping area was detected between the incoming vestibular afferents and trigeminal motoneurons along the whole extent of the trigeminal motor nucleus. The vestibular axon collaterals formed large numbers of close appositions with dorsomedial and ventrolateral dendrites of trigeminal motoneurons. The majority of direct contacts were located on proximal dendritic segments closer than 300µm to the somata. The identified contacts were evenly distributed on rostral motoneurons innervating jaw-closing muscles and motoneurons supplying jaw-opening muscles and located in the caudal part of trigeminal nucleus. We suggest that the identified contacts between vestibular axon terminals and trigeminal motoneurons may constitute one of the morphological substrates of a very quick response detected in trigeminal motoneurons during head movements.

Mesopontine Neurons Implicated in Anesthetic Loss-of-consciousness have Either Ascending or Descending Axonal Projections, but Not Both

The MPTA (mesopontine tegmental anesthesia area) is a key node in a network of axonal pathways that collectively engage the key components of general anesthesia: immobility and atonia, analgesia, amnesia and loss-of-consciousness. In this study we have applied double retrograde tracing to analyze MPTA connectivity, with a focus on axon collateralization. Prior tracer studies have shown that collectively, MPTA neurons send descending projections to spinal and medullary brain targets associated with atonia and analgesia as well as ascending projections to forebrain structures associated with amnesia and arousal. Here we ask whether individual MPTA neurons collateralize broadly as might be expected of modulatory circuitry, sending axonal branches to both caudal and to rostral targets, or whether connectivity is more selective. Two distinguishable retrograde tracers were microinjected into pairs (“dyads”) of known synaptic targets of the MPTA, one caudal and one rostral. We found that neurons that were double-labeled, and hence project to both targets were rare, constituting <0.5% on average of all MPTA neurons that project to these targets. The large majority sent axons either caudally, presumably to mediate mobility and/or antinociception, or rostrally, presumably to mediate mnemonic and/or arousal/cognitive functions. MPTA neurons with descending vs ascending projections also differed in size and shape, supporting the conclusion that they constitute distinct neuronal populations. From these and prior observations we conclude that the MPTA has a hybrid architecture including neurons with heterogeneous patterns of connectivity, some highly collateralized and some more targeted.

Imaging neuronal structure dynamics using 2-photon super-resolution patterned excitation reconstruction microscopy.

Visualizing fine neuronal structures deep inside strongly light-scattering brain tissue remains a challenge in neuroscience. Recent nanoscopy techniques have reached the necessary resolution but often suffer from limited imaging depth, long imaging time or high light fluence requirements. Here, we present two-photon super-resolution patterned excitation reconstruction (2P-SuPER) microscopy for 3-dimensional imaging of dendritic spine dynamics at a maximum demonstrated imaging depth of 130 μm in living brain tissue with approximately 100 nm spatial resolution. We confirmed 2P-SuPER resolution using fluorescence nanoparticle and quantum dot phantoms and imaged spiny neurons in acute brain slices. We induced hippocampal plasticity and showed that 2P-SuPER can resolve increases in dendritic spine head sizes on CA1 pyramidal neurons following theta-burst stimulation of Schaffer collateral axons. 2P-SuPER further revealed nanoscopic increases in dendritic spine neck widths, a feature of synaptic plasticity that has not been thoroughly investigated due to the combined limit of resolution and penetration depth in existing imaging technologies.

Excitatory Synaptic Input to Hilar Mossy Cells under Basal and Hyperexcitable Conditions.

Hilar mossy cells (HMCs) in the hippocampus receive glutamatergic input from dentate granule cells (DGCs) via mossy fibers (MFs) and back-projections from CA3 pyramidal neuron collateral axons. Many fundamental features of these excitatory synapses have not been characterized in detail despite their potential relevance to hippocampal cognitive processing and epilepsy-induced adaptations in circuit excitability. In this study, we compared pre- and postsynaptic parameters between MF and CA3 inputs to HMCs in young and adult mice of either sex and determined the relative contributions of the respective excitatory inputs during in vitro and in vivo models of hippocampal hyperexcitability. The two types of excitatory synapses both exhibited a modest degree of short-term plasticity, with MF inputs to HMCs exhibiting lower paired-pulse (PP) and frequency facilitation than was described previously for MF-CA3 pyramidal cell synapses. MF-HMC synapses exhibited unitary excitatory synaptic currents (EPSCs) of larger amplitude, contained postsynaptic kainate receptors, and had a lower NMDA/AMPA receptor ratio compared to CA3-HMC synapses. Pharmacological induction of hippocampal hyperexcitability in vitro transformed the abundant but relatively weak CA3-HMC connections to very large amplitude spontaneous bursts of compound EPSCs (cEPSCs) in young mice (∼P20) and, to a lesser degree, in adult mice (∼P70). CA3-HMC cEPSCs were also observed in slices prepared from mice with spontaneous seizures several weeks after intrahippocampal kainate injection. Strong excitation of HMCs during synchronous CA3 activity represents an avenue of significant excitatory network generation back to DGCs and might be important in generating epileptic networks.

Mouse subthalamic nucleus neurons with local axon collaterals.

The neuronal population of the subthalamic nucleus (STN) has the ability to prolong incoming cortical excitation. This could result from intra-STN feedback excitation. The combination of inducible genetic fate mapping techniques with in vitro targeted patch-clamp recordings, allowed identifying a new type of STN neurons that possess a highly collateralized intrinsic axon. The time window of birth dates was found to be narrow (E10.5-E14.5) with very few STN neurons born at E10.5 or E14.5. The fate mapped E11.5-12.5 STN neuronal population included 20% of neurons with profuse axonal branching inside the nucleus and a dendritic arbor that differed from that of STN neurons without local axon collaterals. They had intrinsic electrophysiological properties and in particular, the ability to generate plateau potentials, similar to that of STN neurons without local axon collaterals and more generally to that of classically described STN neurons. This suggests that a subpopulation of STN neurons forms a local glutamatergic network, which together with plateau potentials, allow amplification of hyperdirect cortical inputs and synchronization of the STN neuronal population.

FOLATE PREVENTS THE DETRIMENTAL EFFECTS OF OLIGOMERIC Aβ ON INSULIN RECEPTOR LOCALIZATION AND FUNCTION AND LONG-TERM POTENTIATION

Oligomeric Aβ peptide toxicity may contribute to neuronal dysfunction and neurodegeneration in Alzheimer's disease (AD). For example, we showed that Aβ toxicity may underlie AD-associated “Type 3 Diabetes” by inhibiting Kinesin-5, thereby preventing the normal localization and function of the insulin receptor and the glucose transporter (AAIC, 2016). Aβ treatment acutely inhibits long-term potentiation (LTP) and results in synaptic loss, which is mediated in part by Aβ-mediated inhibition of Kinesin-5, likely via mis-localization of key neurotransmitter receptors. In view of evidence that folic acid/folate supplementation may delay/reduce the incidence of AD, we sought to determine whether folate can prevent the toxic effects of Aβ on insulin receptor localization and function and on LTP. N2A cells in culture were co-treated with folate (2 μM) and Aβ42 peptide (2 μM), and insulin receptor localization, function, and phosphorylation were assessed by immunocytochemistry following insulin challenge. LTP in the stratum radiatum was also assessed in mouse brain slices by response to high frequency (2 x 100 Hz) stimulation (HFS) of the Schaffer collateral axons. LTP was strongly inhibited by 100 nM oligomeric Aβ peptide. In the presence of Aβ peptide, folate (2 μM) was added either for 40 min before the HFS (short) or for at least 60 min prior to the HFS and for the remaining 60 min of the recording period (long). N2A cells treated with oligomeric Aβ (48 h) alone exhibited decreased insulin receptor activation (phosphorylation), and the majority of the insulin receptor localized to small aggregates associated with the cis-Golgi, rather to the cell surface. N2A cells co-treated with folate and Aβ maintained normal cell surface localization and function/phosphorylation of the insulin receptor in response to insulin stimulation. ‘Long’ treatment of the mouse brain slices, but not ‘short’ treatment, with folate restored LTP in the presence of oligomeric Aβ. Folate pre-treatment blocks oligomeric Aβ-mediated inhibition of neuronal metabolism and memory function. These results provide a mechanism by which folate can prevent the Aβ toxicity and reinforce the concept that folate supplementation may slow the development of AD.

Nonspecific block of voltage-gated potassium channels has greater effect on distal schaffer collaterals than proximal schaffer collaterals during periods of high activity

Previous studies established different responses between proximal and distal portions of Schaffer collateral axons during high‐frequency and burst stimulation, with distal axons demonstrating biphasic changes in excitability (hyperexcitability followed by depression), but proximal axons showing only monophasic depression. Voltage‐dependent potassium (KV) channels are important determinants of axonal excitability, and block of KV channels can promote axon hyperexcitability. We therefore hypothesized that block of KV channels should lead to biphasic response changes in proximal Schaffer collaterals, like those seen in distal Schaffer collaterals. To test this hypothesis, we made extracellular recordings of distal Schaffer collateral responses in stratum radiatum of hippocampal area CA1 and proximal Schaffer collateral responses in stratum pyramidale of area CA3 during high‐frequency stimulation (HFS) at 100 Hz and burst stimulation at 200 msec intervals (5 Hz or theta frequency). We then applied a nonselective KV channel blocker, tetraethlylammonium (TEA, 10 mmol/L) or 4‐aminopyridine (4‐AP, 100 μmol/L), and assessed effects on Schaffer collateral responses. Surprisingly, block of KV channels had little or no effect on proximal Schaffer collateral responses during high‐frequency or burst stimulation. In contrast, KV channel blockade caused more rapid depression of distal Schaffer collateral responses during both high‐frequency and burst stimulation. These findings indicate that KV channels are important for maintaining distal, but not proximal, Schaffer collateral excitability during period of sustained high activity. Differential sensitivity of distal versus proximal Schaffer collaterals to KV channel block may reflect differences in channel density, diversity, or subcellular localization.

Functional perturbation of forebrain principal neurons reveals differential effects in novel and well-learned tasks

Neural circuits in mammalian brains consist of large numbers of different cell types having different functional properties. To better understand the separate roles of individual neuron types in specific aspects of spatial learning and memory, we perturbed the function of principal neurons in vivo during maze performance or in hippocampal slices during recording of evoked excitatory synaptic potentials. Transgenic mice expressing the Drosophila allatostatin receptor (AlstR) in cortical and hippocampal pyramidal cells were tested on an elevated plus maze, in a Y-maze, and in the Morris water maze. Relative to a control cohort, AlstR-positive mice treated with allatostatin exhibited no difference in open arm dwell time on the elevated plus maze or total number of arm entries in a Y-maze, but displayed reduced spontaneous alternation. When animals received massed or spaced training trials in the Morris water maze, and the peptide was delivered prior to an immediate probe, no effects on performance were observed. When the peptide was delivered during a probe trial performed 24h after seven days of spaced training, allatostatin delivery to AlstR positive mice enhanced direct navigation to the escape platform. Combined, these results suggest that cortical and hippocampal pyramidal neurons are required during spatial decision-making in a novel environment and compete with other neural systems after extended training in a long-term reference memory task. In hippocampal slices collected from AlstR positive animals, allatostatin delivery produced frequency dependent alterations in the Schaffer collateral fiber volley (attenuated accommodation at 100Hz) and excitatory postsynaptic potential (attenuated facilitation at 5Hz). Combined, the neural and behavioral discoveries support the involvement of short-term plasticity of Schaffer collateral axons and synapses during exploration of a novel environment and during initial orientation to a goal in a well-learned setting.

Substance P effects exclusively on prototypic neurons in mouse globus pallidus.

Previous studies have suggested that the neurokinin-1 receptor (NK-1R) expressing neurons in the globus pallidus (GP) receive substance P (SP), presumably released by axon collaterals of striatal direct neurons. However, the effect of SP on the GP remains unclear. In this study, we identified that the SP-responsive cells comprise a highly specific cell type in the GP with regard to immunofluorescence, electrophysiology, and projection properties. Morphologically, NK-1R-immunoreactive neurons occasionally co-expressed parvalbumin (PV) and/or Lim-homeobox 6 (Lhx6), but not Forkhead box protein P2 (FoxP2), which is mainly expressed by arkypallidal neurons. Retrograde tracing experiments also showed that some of GP neurons projecting to the subthalamic nucleus (namely prototypic neurons) expressed NK-1R as well as Lhx6 and/or PV, but not FoxP2. In vitro electrophysiological study revealed that, among 48 GP neurons, the SP agonist induced inward current in 21 neurons. The response was prevented by bath application of the NK-1R antagonist. Based on the firing properties, 92 recorded GP neurons were classified into three distinct types, i.e., CL1, 2, and 3. Interestingly, all the SP-responsive neurons were found to be in CL2 and CL3 types, but not in CL1. Moreover, active and passive membrane properties of the neurons in those clusters and immunofluorescent identification suggested that CL1 and CL2/3 could be considered as arkypallidal and prototypic neurons, respectively. Therefore, SP-responsive neurons were one of the populations of prototypic neurons based on both anatomical and electrophysiological results. Altogether, the striatal direct pathway neurons could affect the indirect pathway in the way of prototypic neurons, via the action of SP to NK-1R.

Synergistic Signaling by Light and Acetylcholine in Mouse Iris Sphincter Muscle

The mammalian pupillary light reflex (PLR) involves a bilateral brain circuit whereby afferent light signals in the optic nerve ultimately drive iris-sphincter-muscle contraction via excitatory cholinergic parasympathetic innervation [1, 2]. Additionally, the PLR in nocturnal and crepuscular sub-primate mammals has a "local" component in the isolated sphincter muscle [3-5], as in amphibians, fish, and bird [6-10]. In mouse, this local PLR requires the pigment melanopsin [5], originally found in intrinsically photosensitive retinal ganglion cells (ipRGCs) [11-19]. However, melanopsin's presence and effector pathway locally in the iris remain uncertain. The sphincter muscle itself may express melanopsin [5], or its cholinergic parasympathetic innervation may be modulated by suggested intraocular axonal collaterals of ipRGCs traveling to the eye's ciliary body or even to the iris [20-22]. Here, we show that the muscarinic receptor antagonist, atropine, eliminated the effect of acetylcholine (ACh), but not of light, on isolated mouse sphincter muscle. Conversely, selective genetic deletion of melanopsin in smooth muscle mostly removed the light-induced, but not the ACh-triggered, increase in isolated sphincter muscle's tension and largely suppressed the local PLR invivo. Thus, sphincter muscle cells are bona fide, albeit unconventional, photoreceptors. We found melanopsin expression in a small subset of mouse iris sphincter muscle cells, with the light-induced contractile signal apparently spreading through gap junctions into neighboring muscle cells. Light and ACh share a common signaling pathway in sphincter muscle. In summary, our experiments have provided details ofa photosignaling process in the eye occurring entirely outside the retina.

Oxytocin: Coevolution of human and domesticated animals

The neuropeptide oxytocin (OT) and its homologs are produced in specialized neurons located in vertebrates exclusively in a deep and evolutionarily old part of the forebrain, the hypothalamus. The axons of OT neurons form the classical hypothalamic-neurohypophyseal tract terminating in the blood vessels of the neurohypothysis to release OT into the system’s blood circulation. However, as was recently demonstrated in mammals, collaterals of OT axons concomitantly project to various forebrain regions to modulate the activity of the local networks. At the behavioral level, OT facilitates intraspecific social contacts in mammals via various mechanisms ranging from the suppression of neuroendocrine stress responses to direct OT action on the neurons of the socially relevant brain regions. Recent reports have indicated the possible contribution of OT to the formation of a social bond between domesticated mammals (dog, sheep, cattle) and humans. Indeed, the social interaction between humans and domesticated animals resulted in the elevation of peripheral OT levels (in blood, saliva, or urine) and, in congruence, exogenous (intranasal) OT application led to more frequent contacts between the owner and the domesticated animal. It has been known for decades that domesticated animals exhibit profound socio-communicative abilities accompanied by suppressed aggression and stress responsiveness. These peculiarities of their behavior and physiology may be influenced by the activity of the central OT system. Therefore, in the present mini review, we focus on the role of OT in the orchestration of distinct forms of social behavior, including the monogamous bond, maternal care, social memory and recognition, aggression, and anxiety. As a conclusion, we propose possible directions for exploring the OT contribution to the empathy between humans and domesticated animals, which was likely established in the course of their coevolution during last 10000–15000 years.

Synaptic Targeting of Double-Projecting Ventral CA1 Hippocampal Neurons to the Medial Prefrontal Cortex and Basal Amygdala

The acquisition and retrieval of contextual fear memory requires coordinated neural activity in the hippocampus, medial prefrontal cortex (mPFC), and amygdala. The contextual information encoded in the hippocampus is conveyed to the mPFC and amygdala for contextual fear conditioning. Previous studies have suggested that a CA1 neuronal population in the ventral hippocampus (VH) projects to both the mPFC and amygdala and is recruited in context-dependent control of conditioned fear. However, how double-projecting ventral CA1 hippocampal (vCA1) neurons modulate the activity of the mPFC and amygdala at the synaptic level has not been determined previously. Here, we show that the optogenetic silencing of the VH prevented the recall of contextual fear memory in mice, indicating its role in contextual fear expression. In dual retrograde viral tracing and c-Fos immunostaining experiments, we found that a proportion of vCA1 neurons projected to both the mPFC and amygdala and were recruited preferentially during context exposure, suggesting their role in encoding context representations. Moreover, optogenetic stimulation of axon collaterals of double-projecting vCA1 neurons induced monosynaptic excitatory responses in both the mPFC and basal amygdala, indicating that they could convey contextual information through the VH-mPFC and VH-amygdala pathways. The activation of double-projecting vCA1 neurons also induced action potential firings in the mPFC neurons that project to the amygdala, suggesting that they can also activate the VH-mPFC-amygdala pathway. With these synaptic mechanisms, double-projecting vCA1 neurons could induce synchronized neural activity in the mPFC and amygdala and convey contextual information efficiently to the basal amygdala for contextual fear conditioning.SIGNIFICANCE STATEMENT This work demonstrates that ventral CA1 hippocampal (vCA1) neurons projecting to both the medial prefrontal cortex (mPFC) and amygdala are activated preferentially when contextual information is processed in the ventral hippocampus, which is required for contextual fear expression. Our electrophysiological experiments reveal that the activation of double-projecting vCA1 neurons induces excitatory synaptic activity in both the mPFC and amygdala. These results suggest that double-projecting vCA1 neurons could contribute to contextual fear responses by inducing synchronized activity in the mPFC and amygdala and conveying contextual information to the basal amygdala more efficiently than vCA1 neurons projecting to either the mPFC or amygdala alone. These findings provide important insights into the mechanisms of the acquisition and retrieval of contextual fear memory.

Pursuit of Neurotransmitter Functions: Being Attracted with Fascination of the Synapse.

In the beginning of the 1970s, only two chemical substances, acetylcholine and γ-aminobutyric acid (GABA), had been definitely established as neurotransmitters. Under such circumstances, I started my scientific career in Professor Masanori Otsuka's lab searching for the transmitter of primary sensory neurons. Until 1976, lines of evidence had accumulated indicating that the undecapeptide substance P could be released as a transmitter from primary afferent fibers into spinal synapses, although the substance P-mediated synaptic response had yet to be identified. Peripheral synapses could serve as a good model and thus, it was demonstrated in the prevertebral sympathetic ganglia by1985 that substance P released from axon collaterals of primary sensory neurons acts as the transmitter mediating non-cholinergic slow excitatory postsynaptic potential (EPSP). At that time, we also found that autonomic synapses were useful to uncover the transmitter role of the opioid peptide enkephalins, whose functions had been unknown since their discovery in 1975. Accordingly, enkephalins were found to serve a transmitter role in mediating presynaptic inhibition of cholinergic fast and non-cholinergic slow transmission in the prevertebral sympathetic ganglia. In 1990s, we attempted to devise a combined technique of brain slices and patch-clamp recordings. We applied it to study the regulatory mechanisms that operate around cerebellar GABAergic inhibitory synapses, because most of the studies then had centered on excitatory synapses and because inhibitory synapses are crucially involved in brain functions and disorders. Consequently, we discovered novel forms of heterosynaptic interactions, dual actions of a single transmitter, and receptor crosstalk, the details of which are described in this review.

A retinoraphe projection regulates serotonergic activity and looming-evoked defensive behaviour

Animals promote their survival by avoiding rapidly approaching objects that indicate threats. In mice, looming-evoked defensive responses are triggered by the superior colliculus (SC) which receives direct retinal inputs. However, the specific neural circuits that begin in the retina and mediate this important behaviour remain unclear. Here we identify a subset of retinal ganglion cells (RGCs) that controls mouse looming-evoked defensive responses through axonal collaterals to the dorsal raphe nucleus (DRN) and SC. Looming signals transmitted by DRN-projecting RGCs activate DRN GABAergic neurons that in turn inhibit serotoninergic neurons. Moreover, activation of DRN serotoninergic neurons reduces looming-evoked defensive behaviours. Thus, a dedicated population of RGCs signals rapidly approaching visual threats and their input to the DRN controls a serotonergic self-gating mechanism that regulates innate defensive responses. Our study provides new insights into how the DRN and SC work in concert to extract and translate visual threats into defensive behavioural responses.

The role of axonal Kv1 channels in CA3 pyramidal cell excitability

Axonal ion channels control spike initiation and propagation along the axon and determine action potential waveform. We show here that functional suppression of axonal Kv1 channels with local puff of dendrotoxin (DTx), laser or mechanical axotomy significantly increased excitability measured in the cell body. Importantly, the functional effect of DTx puffing or axotomy was not limited to the axon initial segment but was also seen on axon collaterals. In contrast, no effects were observed when DTx was puffed on single apical dendrites or after single dendrotomy. A simple model with Kv1 located in the axon reproduced the experimental observations and showed that the distance at which the effects of axon collateral cuts are seen depends on the axon space constant. In conclusion, Kv1 channels located in the axon proper greatly participate in intrinsic excitability of CA3 pyramidal neurons. This finding stresses the importance of the axonal compartment in the regulation of intrinsic neuronal excitability.

Monoaminergic modulation of GABAergic transmission onto cerebellar globular cells

Cerebellar Purkinje cells (PCs) project their axon collaterals to underneath of the PC layer and make GABAergic synaptic contacts with globular cells, a subgroup of Lugaro cells. GABAergic transmission derived from the PC axon collaterals is so powerful that it could inhibit globular cells and regulate their firing patterns. However, the physiological properties and implications of the GABAergic synapses on globular cells remain unknown. Using whole-cell patch-clamp recordings from globular cells in the mouse cerebellum, we examined the monoaminergic modulation of GABAergic inputs to these cells. Application of either serotonin (5-HT) or noradrenaline (NA) excited globular cells, thereby leading to their firing. The 5-HT- and NA-induced firing was temporally confined and attenuated by GABAergic transmission, although 5-HT and NA exerted an inhibitory effect on the release of GABA from presynaptic terminals of PC axon collaterals. Agonists for 5-HT1B receptors and α2-adrenoceptors mimicked the 5-HT- and NA-induced suppression of GABAergic activity. Through their differential modulatory actions on the cerebellar inhibitory neural circuits, 5-HT facilitated PC firing, whereas NA suppressed it. These results indicate that 5-HT and NA regulate the membrane excitability of globular cells and PCs through their differential modulation of not only the membrane potential but also GABAergic synaptic circuits. Monoaminergic modulation of the neural connections between globular cells and PCs could play a role in cerebellar motor coordination.

Loss of Saltation and Presynaptic Action Potential Failure in Demyelinated Axons

In cortical pyramidal neurons the presynaptic terminals controlling transmitter release are located along unmyelinated axon collaterals, far from the original action potential (AP) initiation site, the axon initial segment (AIS). Once initiated, APs will need to reliably propagate over long distances and regions of geometrical inhomogeneity like branch points (BPs) to rapidly depolarize the presynaptic terminals and confer temporally precise synaptic transmission. While axon pathologies such as demyelinating diseases are well established to impede the fidelity of AP propagation along internodes, to which extent myelin loss affects propagation along BPs and axon collaterals is not well understood. Here, using the cuprizone demyelination model, we performed optical voltage-sensitive dye (VSD) imaging from control and demyelinated layer 5 pyramidal neuron axons. In the main axon, we find that myelin loss switches the modality of AP propagation from rapid saltation towards a slow continuous wave. The duration of single AP waveforms at BPs or nodes was, however, only slightly briefer. In contrast, by using two-photon microscopy-guided loose-seal patch recordings from axon collaterals we revealed a presynaptic AP broadening in combination with a reduced velocity and frequency-dependent failure. Finally, internodal myelin loss was also associated with de novo sprouting of axon collaterals starting from the primary (demyelinated) axon. Thus, the loss of oligodendrocytes and myelin sheaths bears functional consequences beyond the main axon, impeding the temporal fidelity of presynaptic APs and affecting the functional and structural organization of synaptic connectivity within the neocortex.

Quantitative analysis of axon collaterals of single pyramidal cells of the anterior piriform cortex of the guinea pig

BackgroundThe role of the piriform cortex (PC) in olfactory information processing remains largely unknown. The anterior part of the piriform cortex (APC) has been the focus of cortical-level studies of olfactory coding, and associative processes have attracted considerable attention as an important part in odor discrimination and olfactory information processing. Associational connections of pyramidal cells in the guinea pig APC were studied by direct visualization of axons stained and quantitatively analyzed by intracellular biocytin injection in vivo.ResultsThe observations illustrated that axon collaterals of the individual cells were widely and spatially distributed within the PC, and sometimes also showed a long associational projection to the olfactory bulb (OB). The data showed that long associational axons were both rostrally and caudally directed throughout the PC, and the intrinsic associational fibers of pyramidal cells in the APC are omnidirectional connections in the PC. Within the PC, associational axons typically followed rather linear trajectories and irregular bouton distributions. Quantitative data of the axon collaterals of two pyramidal cells in the APC showed that the average length of axonal collaterals was 101 mm, out of which 79 mm (78% of total length) were distributed in the PC. The average number of boutons was 8926 and 7101, respectively, with 79% of the total number of boutons being distributed in the PC. The percentage of the total area of the APC and the posterior piriform cortex occupied by the average distribution region of the axon collaterals of two superficial pyramidal (SP) cells was about 18 and 5%, respectively.ConclusionOur results demonstrate that omnidirectional connection of pyramidal cells in the APC provides a substrate for recurrent processes. These findings indicate that the axon collaterals of SP cells in the PC could make synaptic contacts with all granule cells in the OB. This study provides the morphological evidence for understanding the mechanisms of information processing and associative memory in the APC.

Furosemide depresses the presynaptic fiber volley and modifies frequency-dependent axonal excitability in rat hippocampus.

The loop diuretic furosemide is known to have anticonvulsant effects, believed to be exerted through blockade of glial Na+-K+-2Cl- cotransport causing altered volume regulation in brain tissue. The possibility that direct effects of furosemide on neuronal properties could also be involved is supported by previous observations, but such effects have not been thoroughly investigated. In the present study we show that furosemide has two opposing effects on stimulus-induced postsynaptic excitation in the nonepileptic rat hippocampal slice: 1) an enhancement of e-s coupling, which depended on intact GABAA transmission and was partially mimicked by selective blockade of K+-2Cl- cotransport, and 2) a decrement of field excitatory postsynaptic potentials. The balance between these effects varied, depending on the amount of synaptic drive. In addition, the compound action potential (fiber volley) recorded from the stimulated Schaffer collateral axons in stratum radiatum showed a progressive decrease during perfusion of furosemide. This effect was activity-independent, was mimicked by the stilbene derivative DIDS, and could be reproduced on fiber volleys in the alveus. Furosemide also reduced the initial enhancement of the fiber volley observed during trains of high-frequency stimulation (HFS). Results of hyperosmotic expansion of the extracellular volume, with 30 mM sucrose, indicated that both the induction and antagonism of the HFS-induced enhancement were independent of signaling via the extracellular space. Furosemide caused an increased decay of paired-pulse-induced supranormal axonal excitability, which was antagonized by ZD7288. We conclude that furosemide decreases axonal excitability and prevents HFS-induced hyperexcitability via mechanisms downstream of blockage of anion transport, which could include hyperpolarization of axonal membranes.NEW & NOTEWORTHY This study shows that the anion transporter antagonists furosemide and DIDS cause a marked decrease of axonal excitability in rat hippocampal CA1 region and prevent the induction of activity-dependent hyperexcitability in Schaffer collateral axons. The data are consistent with direct effects on axonal membrane properties. We also find that activity-dependent enhancement and depression of axonal excitability can be modified independently, suggesting that these events are governed by different underlying processes.