The Slow Depolarization Following Individual Spikes in Thin, Unmyelinated Axons in Mammalian Cortex.

An important goal in neuroscience is to understand how neuronal excitability is controlled. Therefore, Gardner-Medwin's 1972 discovery, that cerebellar parallel fibers were more excitable up to 100 ms after individual action potentials, could have had great impact. If this long-lasting effect were due to intrinsic membrane mechanisms causing a depolarizing after-potential (DAP) this was an important finding. However, that hypothesis met resistance because the use of K+ sensitive electrodes showed that synchronous activation, as commonly used in excitability tests, increased extracellular K+ concentration sufficiently to explain much of the hyperexcitability. It is still controversial because intra-axonal recordings, which could have settled the debate, have not been made from parallel fibers or other axons of similar calibers. If it had not been for the fact that such thin axons are, by far, the most common axon type in cortical areas and control almost all glutamate release, it would be tempting to ignore them until an appropriate intra-axonal recording technique is invented. I will go through the literature that, taken together, supports the hypothesis that a DAP is an intrinsic membrane mechanism in cerebellar parallel fibers and hippocampal Schaffer collaterals. It is most likely due to a well-controlled process that stops the fast repolarization at a membrane potential positive to resting membrane potential, leaving the membrane more excitable for ~100 ms during a slow, passive discharge of the membrane capacitance. The DAP helps reduce failures but can also cause uncontrolled bursting if it is not properly controlled. The voltage at which the fast repolarization stops, and the DAP starts, is close the activation range of both Na+ and Ca2+ voltage activated channels and is therefore essential for neuronal function.

Typical gray matter axons in mammalian brain fail to conduct action potentials faithfully at fever-like temperatures.

We studied the ability of typical unmyelinated cortical axons to conduct action potentials at fever-like temperatures because fever often gives CNS symptoms. We investigated such axons in cerebellar and hippocampal slices from 10 to 25 days old rats at temperatures between 30 and 43°C. By recording with two electrodes along axonal pathways, we confirmed that the axons were able to initiate action potentials, but at temperatures >39°C, the propagation of the action potentials to a more distal recording site was reduced. This temperature-sensitive conduction may be specific for the very thin unmyelinated axons because similar recordings from myelinated CNS axons did not show conduction failures. We found that the conduction fidelity improved with 1 mmol/L TEA in the bath, probably due to block of voltage-sensitive potassium channels responsible for the fast repolarization of action potentials. Furthermore, by recording electrically activated antidromic action potentials from the soma of cerebellar granule cells, we showed that the axons failed less if they were triggered 10-30 msec after another action potential. This was because individual action potentials were followed by a depolarizing after-potential, of constant amplitude and shape, which facilitated conduction of the following action potentials. The temperature-sensitive conduction failures above, but not below, normal body temperature, and the failure-reducing effect of the spike's depolarizing after-potential, are two intrinsic mechanisms in normal gray matter axons that may help us understand how the hyperthermic brain functions.

Components of action potential repolarization in cerebellar parallel fibres.

Repolarization of the presynaptic action potential is essential for transmitter release, excitability and energy expenditure. Little is known about repolarization in thin, unmyelinated axons forming en passant synapses, which represent the most common type of axons in the mammalian brain's grey matter.We used rat cerebellar parallel fibres, an example of typical grey matter axons, to investigate the effects of K(+) channel blockers on repolarization. We show that repolarization is composed of a fast tetraethylammonium (TEA)-sensitive component, determining the width and amplitude of the spike, and a slow margatoxin (MgTX)-sensitive depolarized after-potential (DAP). These two components could be recorded at the granule cell soma as antidromic action potentials and from the axons with a newly developed miniaturized grease-gap method. A considerable proportion of fast repolarization remained in the presence of TEA, MgTX, or both. This residual was abolished by the addition of quinine. The importance of proper control of fast repolarization was demonstrated by somatic recordings of antidromic action potentials. In these experiments, the relatively broad K(+) channel blocker 4-aminopyridine reduced the fast repolarization, resulting in bursts of action potentials forming on top of the DAP. We conclude that repolarization of the action potential in parallel fibres is supported by at least three groups of K(+) channels. Differences in their temporal profiles allow relatively independent control of the spike and the DAP, whereas overlap of their temporal profiles provides robust control of axonal bursting properties.

Action potentials recorded from bundles of very thin, gray matter axons in rat cerebellar slices using a grease-gap method.

We investigated the ability of a grease-gap method to record fast and slow changes of the membrane potential from bundles of gray matter axons. Their membrane potentials are of particular interest because these axons are different from most axons that have been investigated using intra-axonal or gap techniques. One of the main differences is that gray matter axons typically have closely spaced presynaptic specializations, called boutons or varicosities, distributed along their entire paths. In response to electrical activation of bundles of parallel fiber axons we were able to record small (128-416µV) but stable signals that we show most likely represented a fraction of the trans-membrane action potentials. A less-than 100% fraction prevents measurements of absolute values for membrane potentials, but the good signal-to-noise ratio (typically 10-16) allows detection of changes in resting membrane potential, action potentials and their after-potentials. Because very little is known about the shape of action potentials and after-potentials in these axons we used several independent methods to make it likely that the grease-gap signal was of intra-axonal origin. We demonstrate the utility of the method by showing that the action potentials in cerebellar parallel fibers and hippocampal Schaffer collaterals had a slowly decaying, depolarized after-potential. The method is ideal for pharmacological tests, which we demonstrate by showing that the slow after-potential was sensitive to 4-AP, and that the membrane potential was reduced by 200µM Ba(2+).

The influence of orexins on the firing rate and pattern of rat intergeniculate leaflet neurons--electrophysiological and immunohistological studies.

Orexins influence various physiological processes associated with feeding behaviour, endocrine functions and wakefulness. One component of mammalian circadian timing systems, intergeniculate leaflet (IGL) of the lateral geniculate nucleus, is thought to contribute to circadian entrainment by processing photic and non-photic/arousal-related signals. Because the IGL is possibly innervated by the orexinergic system, using in vitro extracellular recording techniques we evaluated the influence of orexin A (OXA) and orexin B (OXB) on the rate and pattern of neuronal firing in this structure. Significant increases in the activity of 33 and 28% of IGL cells were observed after locally applied OXA (1 µm) and OXB (1 µm), respectively. In the great majority of neurons responses to OXA were maintained in the presence of orexin-1 receptor OX1R antagonist, SB 334867 (10 µm). Additionally, 75% of the OXB-responsive neurons were also sensitive to an orexin-2 receptor (OX2R)-selective agonist, [Ala11, D-Leu15]-OXB (1 µm). Immunohistochemical stainings showed putative synaptic contacts between OXA- and OXB-immunoreactive fibres and neuropeptide Y, and enkephalin-positive neurons in the investigated area. The outcome of our experiments reinforces previous reports indicating the possible linkage between the orexinergic and circadian systems. To our knowledge the presented findings are the first showing the direct influence of orexins on the IGL activity, mostly through activation of OX2R.

The H-current secures action potential transmission at high frequencies in rat cerebellar parallel fibers.

Most axons in the mammalian brain are unmyelinated and thin with pre-synaptic specializations (boutons) along their entire paths. The parallel fibers in the cerebellum are examples of such axons. Unlike most thin axons they have only one branch point. The granule cell soma, where they originate, can fire bursts of action potentials with spike intervals of about 2 ms. An important question is whether the axons are able to propagate spikes with similarly short intervals. By using extracellular single-unit and population-recording methods we showed that parallel fibers faithfully conduct spikes at high frequencies over long distances. However, when adding 20 microm ZD7288 or 1 mm Cs(+), or reducing the temperature from 35 to 24 degrees C, the action potentials often failed even when successfully initiated. Ba(2+)(1 mm), which blocks Kir channels, did not reproduce these effects. The conduction velocity was reduced by ZD7288 but not by Ba(2+). This suggests that the parallel fibers have an H-current that is active at rest and that is important for their frequency-following properties. Interestingly, failures occurred only when the action potential had to traverse the axonal branch point, suggesting that the branch point is the weakest point in these axons.

Contribution of morphology and membrane resistance to integration of fast synaptic signals in two thalamic cell types.

Thalamocortical cells (TCs) and interneurons (INs) in the lateral geniculate nucleus process visual information from the retina. The TCs have many short dendrites, whereas the INs have fewer and longer dendrites. Because of these morphological differences, it has been suggested that transmission of synaptic signals from dendritic synapses to soma is more efficient in TCs than in INs. However, a higher membrane resistance (R(m)) for the INs could, in theory, compensate for the attenuating effect of their long dendrites and allow distal synaptic inputs to significantly depolarize the soma. Compartmental models were made from biocytin filled TCs (n = 15) and INs (n = 3) and adjusted to fit the current- and voltage-clamp recordings from the individual cells. The confidence limits for the passive electrical parameters were explored by simulating the influence of noise, morphometric errors and non-uniform and active conductances. One of the useful findings was that R(m) was accurately estimated despite realistic levels of active conductance. Simulations to explore the somatic influence of dendritic synapses showed that a small (0.5 nS) excitatory synapse placed at different dendritic positions gave similar somatic potentials in the individual TCs, within the TC population and also between TCs and INs. A linear increase in the conductance of the synapse gave increases in somatic potentials that were more sublinear in INs than TCs. However, when the total synaptic conductance was increased by simultaneously activating many small, spatially distributed synapses, the INs converted the synaptic signals to soma potentials almost as efficiently as the TCs. Thus, INs can transfer fast synaptic signals to soma as efficiently as TCs except when the focal conductance is large.

Axonal varicosity distributions along parallel fibers: a new angle on a cerebellar circuit.

Presynaptic terminals occur along unmyelinated axons in specialized compartments called axonal varicosities or synaptic boutons. Since the first descriptions of varicose axons by Cajal and others, the spatial organization of varicosities along axons has attracted the attention of neuroscientists. Quantitative light- and electron-microscopic analyses of varicosity spacing in the cerebellum and elsewhere have recently provided a clearer picture of this organization, and theoretical analyses now incorporate varicosity spacing as an essential parameter in structural models of neural connectivity. Here we review the salient features of varicosity spacing, with emphasis on cerebellar parallel fibers as a model system. Measured globally across the entire approximately 5 mm lengths of parallel fibers, the overall mean spacing of varicosities is 5.2 microm. Measured locally, however, mean spacing follows a proximodistal gradient, increasing with distance from the point of bifurcation from the ascending axon. Measured at the level of individual varicosities, parallel fiber varicosity distributions follow a distinct pattern characterized by a fixed relationship between the spacing variability and mean. This pattern equally describes varicosity distributions in a number of other brain regions, and therefore appears to constitute a general scaling relationship for excitatory varicose axons. We further discuss evidence for common principles underlying the placement of both varicosities and synapses along axons.

Single-axon action potentials in the rat hippocampal cortex.

Whether all action potentials propagate faithfully throughout axon arbors in the mammalian CNS has long been debated, and remains an important issue because many synapses occur far from the soma along extremely thin, unmyelinated, varicosity-laden branches of axon arbors. We detected unitary action potentials along individual axon branches of adult hippocampal CA3 pyramidal cells using extracellular electrodes, and analysed their conduction across long distances (mean, 2.1 mm) at 22 and 37 degrees C. Axons nearly always transmitted low-frequency impulses. At higher frequencies, most axons also transmitted impulses with striking fidelity. However, at paired-pulse frequencies in the hundreds of kilohertz range, axons exhibited variability: refractory periods ranged from 2.5 to 10 ms at 37 degrees C and from 5 to 40 ms at 22 degrees C. Although the basis for the refractory period variability could not be determined, these limits overlap with CA3 spike frequencies observed in vivo, raising the possibility that some axonal branches act as filters for the higher-order spikes in bursts, in contrast to the observed first-spike reliability. These results extend the observations of propagation reliability to a much longer distance and higher frequency domain than previously reported, and suggest a high safety factor for action potential propagation along thin, varicose axons.

Conduction latency along CA3 hippocampal axons from rat.

Relatively few physiological studies have been carried out on intrahippocampal axons. We have recorded compound potentials from fiber groups and the activity of individual axons at 22-25 degrees C to characterize the conduction in subsets of the broad fan-shaped CA3 pyramidal axonal tree, including the Schaffer collaterals and longitudinal branches. The same wide axonal branching was indicated by antidromic activation of individual CA3 pyramidal cells. The average compound action potential latency from the CA3 to the CA1 area (Schaffer collaterals) increased by 4.16 +/- 0.06 ms/mm separation between the stimulation and registration electrodes. The impulses spread 31% faster in the 45-degree oblique temporal than in the transverse direction across CA1. The latency of the longitudinal axons in the CA3 area increased by 6.19 +/- 0.19 ms/mm. More impressive than these direction-dependent differences in latency were the large differences between individual axons running in the same direction. For both the longitudinal axons and the Schaffer collaterals, there was a broad distribution of antidromic latencies for a given distance between the stimulation and recording points. Typically, the fastest impulses arrived in half the time of the slowest. The distribution of compound action potential latencies between two points in the tissue could be made narrower by surgical restriction of the thickness and width of the preparation. By comparison, the cerebellar parallel fibers showed a narrower distribution of their latencies than the Schaffer collaterals. Because the cerebellar fibers run more straight than Schaffer collaterals, this suggested that some of the latency differences of the latter were due to differences in the path length of the axons. One consequence of our findings is that synchronous firing of neighboring CA3 pyramidal cells does not necessarily give synchronous inputs to common target CA1 neurons.

An estimator for the electrotonic size of neurons independent of charge equalization time constants.

Electrotonic properties are important aspects of neuronal function but have been difficult to estimate without accurate morphological reconstruction. The complexity of the branching dendritic cables often gives charging curves composed of a very large number of exponential functions, making it difficult to distinguish the time constants that are needed for electrotonic estimates. We describe an estimator P for the electrotonic size of neurons based on simple measures from voltage and current clamp recordings that does not rely on the higher rank exponential components of the response. Our estimator gives a bounded scale for the electrotonic size of the cell and can be used for categorization and comparison when morphology is not available.

General and variable features of varicosity spacing along unmyelinated axons in the hippocampus and cerebellum.

Along unmyelinated central axons, synapses occur at focal swellings called axonal varicosities (boutons). The mechanisms regulating how frequently synapses and varicosities occur along axons remain poorly understood. Here, to investigate varicosity distribution patterns and the extent to which they may be conserved across different axons, we analyzed varicosity numbers and positions along fluorescently labeled axon branches in hippocampal area CA1 (CA3-to-CA1 "Schaffer collateral" axons) and five other synaptic regions of rat hippocampus and cerebellum. Varicosity spacing varied by region; e.g., 3.7 +/- 0.6 microm (mean +/- SD) for CA3-to-CA1 axons and 5.2 +/- 1.0 microm for cerebellar parallel fibers. Surprisingly, when 56 axons from these different regions were pooled into a single heterogeneous group, a general relationship emerged: the spacing variability (SD) was a constant fraction of the mean spacing, suggesting that varicosities along different axons are distributed in a fundamentally similar, scaled manner. Varicosity spacing was neither regular nor random but followed a pattern consistent with random synaptic distributions and the occurrence of multiple-synapse boutons. A quantitative model reproduced the salient features of the data and distinguished between two proposed mechanisms relating axonal morphogenesis and synaptogenesis.

Putative Single Quantum and Single Fibre Excitatory Postsynaptic Currents Show Similar Amplitude Range and Variability in Rat Hippocampal Slices.

Transmission at excitatory synapses in the mammalian brain is thought to depend on the release of transmitter quanta through exocytosis of presynaptic vesicles (Katz, 1969). The number of vesicles released by a single presynaptic action potential is important for understanding the impact of a single synapse, and the variability in transmission from one impulse to the next. In addition, the number of vesicles released may be an important factor for synaptic regulation and plasticity, such as facilitation, post-tetanic potentiation and long-term potentiation (LTP). Three recent studies suggest that an increase in the number of transmitter quanta underlies hippocampal LTP (Malinow and Tsien, 1990; Bekkers and Stevens 1990; Malinow, 1991), whereas other reports suggest a postsynaptic mechanism (Kauer et al., 1988; Muller et al., 1988; Foster and McNaughton, 1989). We have used the whole-cell recording technique to compare putative quantal and single fibre responses at excitatory synapses in rat hippocampal slices, and find (i) a surprisingly large variability in single fibre excitatory postsynaptic currents (sfEPSCs); (ii) an equally large variability of putative quantal (pq) EPSCs elicited by hyperosmolar media or ruthenium red; (iii) the observed amplitude ranges for the sfEPSCs and the pqEPSCs overlap almost completely; and (iv) in neither case can the variability be attributed to a scatter in electrotonic distance from the soma of the engaged synapses. Thus, the data are compatible with the hypothesis that a presynaptic action potential usually releases only a single quantum. Other possibilities are also discussed.