Intracellular recording and staining of neurons in the pigeon nucleus lentiformis mesencephali

The pretectal nucleus lentiformis mesencephali in pigeons is involved in optokinetic nystagmus and consists of lateral (nLMl) and medial (nLMm) subnuclei. The present study using intracellular recordings and brain slices shows that pretectal cells respond to depolarizing current injection in different ways. Type I cells (32%) fire spontaneously and have regular spikes. Type II cells (20%) discharge regular spikes, whose frequency increases as current intensity increases. Type III cells (8%) produce a series of bursts, each of which consists of 2-5 spikes depending on current intensities. Type IV cells (39%) fire several spikes in a cluster at the onset of current injection and are then rapidly adapted. One cell of type V (1%) shows spontaneous firing and is inactivated by depolarizing currents. Cells of types III and V are only found in nLMm, and other types of cells exist in both subnuclei. This physiological difference might be a bias due to the small sampling of cells. Twenty-six cells are labeled with dye and they could be categorized into fusiform (23.1%), piriform (7.7%), or multipolar (69.2%) cells. Some correlation seems to exist between the physiological and morphological properties of pretectal neurons. Statistically, the somatic size of nLMm cells is significantly larger than that of nLMl cells, indicating that the nucleus could be divided cytoarchitecturally into magnocellular and parvocellular components as suggested previously.     

Discharge patterns evoked by depolarizing current injection in basal optic nucleus neurons of the pigeon

The nucleus of the basal optic root of the accessory optic system in birds is involved in optokinetic nystagmus, which stabilizes images on the retina by compensatory movements of the eyes. The present paper studies the physiological and morphological properties of basal optic neurons in the pigeon by using a brain slice preparation and intracellular recordings. Sixty-one cells examined could be categorized into six types based on their firing patterns in response to depolarizing current injection. Type I cells (54%) fire spontaneously and more spikes as current intensity is increased. Type II cells (15%) discharge regular spikes with similar interspike intervals. Type III cells (5%) show an early burst followed by tonic firing. Type IV cells (5%) fire regular bursts with similar interburst intervals. Type V cells (16%) fire a few spikes in a cluster only at onset of current application. Type VI cells (5%) produce a hump-like depolarization or a single spike depending on current intensities. Seventeen cells stained with Lucifer yellow have multipolar or piriform perikarya (15-28 microm) with two to eight primary dendrites. In some cases, an axon is observed to originate from the cell body, traveling dorsolaterally or dorsally. The physiological significance of these findings is discussed.     

Classification of extracellularly recorded neurons by their discharge patterns and their correlates with intracellularly identified neuronal types in the frontal cortex of behaving monkeys

Neurons in the cerebral cortex are not homogeneous. However, neuronal types have been ignored in most previous work studying neuronal processes in behaving monkeys. We propose a new method to identify neuronal types in extracellular recording studies of behaving monkeys. We classified neurons as either bursting or non‐bursting, and then classified the bursting neurons into three types: (i) neurons displaying a burst of many spikes (maximum number of spikes within a burst; NSB max ≥ 8) at a high discharge rate (maximum interspike interval; ISI max < 5 ms); (ii) neurons displaying a burst of fewer spikes (NSB max ≤ 5) at a high discharge rate (ISI max < 5 ms); and (iii) neurons displaying a burst of a few spikes (NSB max ≤ 7) at relatively long ISIs (ISI max > 5 ms). We found that the discharge patterns of the four groups corresponded to those of regular spiking (RS), fast spiking (FS), fast rhythmic bursting (FRB) and intrinsic bursting (IB) neurons demonstrated in intracellular recording studies using in vitro slice preparations, respectively. In addition, we examined correlations with the task events for neurons recorded in the frontal eye field and neuronal interactions for pairs of neurons recorded simultaneously from a single electrode. We found that they were substantially different between RS and FS types. These results suggest that neurons in the frontal cortex of behaving monkeys can be classified into four types based on their discharge patterns, and that these four types contribute differentially to cortical operations.     

Synaptic excitability of the burst firing neurons in cat sensorimotor cortex in vitro

We have recently reported that the burst firing neurons are found in layer III as well as in layer V of cat sensorimotor cortex in vitro. In the present study, we examined the synaptic excitability of layer III neurons by white matter stimulation and compared with their firing patterns against the current injections through the recording microelectrodes. The firing patterns of layer III neurons were classified into three main classes as in our previous study, i.e., (1) regular spiking (RS), i.e., the tonic firing that often exhibited spike-frequency adaptation, (2) burst-and-single spiking (BS), i.e., the initial bursting followed by tonic firing, (3) repetitive-bursting (RB), the burst firing that recurred at fast frequency. In RS cells, single action potential was superimposed on the largest EPSPs among all cell types analyzed. BS cells also fired single action potential and never exhibited burst firing synaptically. Only in a part of RB cells, synaptic bursting instead of single action potential was evoked on smaller EPSPs. IPSPs could be observed in about 60% of all the recorded RS and BS cells, however, they were observed in only 10% of the RB cells.     

Burst generating and regular spiking layer 5 pyramidal neurons of rat neocortex have different morphological features

Intracellular recordings were obtained from pyramidal neurons in layer 5 of rat somatosensory and visual cortical slices maintained in vitro. When directly depolarized, one subclass of pyramidal neurons had the capacity to generate intrinsic burst discharges and another generated regular trains of single spikes. Burst responses were triggered in an all-or-none manner from depolarizing afterpotentials in most bursting neurons. Regular spiking cells responded to electrical stimulation of ascending afferents with a typical EPSP-IPSP sequence, whereas IPSPs were hard to detect in bursting cells. Orthodromic activation of the latter evoked a prominent voltage-dependent depolarization that could trigger a burst response. Intracellularly labelled bursting and regular spiking cells were located in layer 5b, but had distinctly different morphologies. Bursting neurons had a large pyramidal soma, a gradually emerging apical dendrite, and an extensive apical and basal dendritic tree. Their axonal collateral arborization was predominantly limited to layers 5/6. In contrast, regular spiking cells had a more rounded soma with abruptly emerging apical dendrite, a smaller dendritic arborization, and 2 to 8 ascending axonal collaterals that arborized widely in the supragranular layers. Both bursting and regular spiking cells had main axons that entered the subcortical white matter. These data show that some subgroups of pyramidal neurons within the deeper parts of layer 5 of rat cortex are morphologically and physiologically distinct and have different intracortical connections. Bursting cells presumably function to amplify and synchronize cortical outputs, whereas regular spiking output neurons provide excitatory feedback to neurons at all cortical levels and receive a more effective orthodromic inhibitory input. These data support the hypothesis that differences in gross neuronal structure, perhaps even the subtle differences that distinguish subclasses of neurons in a given lamina, are predictive of underlying differences in the type and distribution of ion channels in the nerve cell membrane and connections of cells within the cortical circuit.     

Membrane properties and discharge characteristics of guinea pig dorsal cochlear nucleus neurons studied in vitro

Intracellular recordings were made from neurons of the guinea pig dorsal cochlear nucleus in an in vitro brain slice preparation. The membrane properties of the cells were studied, and the membrane potentials were manipulated by current injection to determine how intrinsic conductances might alter the cell discharge patterns. Eleven cells were marked with Lucifer yellow. Ten of these cells were identified as the large pyramidal cells of layer 2 of this nucleus, and 1 cell was identified as a "vertical" cell in layer 3. Two kinds of action potentials were observed: simple spikes and complex spikes. This report discusses only cells with simple spikes. Simple spiking cells (60/72 recorded cells; all stained cells were simple spiking cells) discharged in a regular fashion with depolarization, and had linear frequency-current relationships up to 2 nA with a mean slope of 116 Hz/nA. The discharge rate was approximately constant throughout the current pulse. Responses of simple spiking cells to depolarizing current steps superimposed on a steady-state membrane hyperpolarization were studied. When the membrane has been held hyperpolarized, small current pulses produce a long-latency regular train of action potentials. Larger current pulses superimposed on membrane hyperpolarization can produce a short-latency action potential followed by a long silent interval (i.e., a long first interspike interval), and finally a regular train of spikes. It is concluded that the membrane conductances of DCN pyramidal cells are capable of generating at least 3 discharge patterns (regular firing, long first spike latency, and long first interspike interval) depending on the state of the membrane potential prior to a depolarizing current step. These responses are similar to the "chopper," "buildup," and "pauser" discharge patterns reported for these cells in vivo in response to tone bursts. The modulation of the intrinsic membrane conductances by membrane polarization and the possible contribution of these conductances to the generation of DCN discharge patterns provide new insights into the mechanisms underlying the responses of DCN cells to acoustic stimuli.     

Comparative distribution of the mammalian mediator subunit thyroid hormone receptor-associated protein (TRAP220) mRNA in developing and adult rodent brain

Disinhibition reliably induces regular synchronous bursting in networks of spinal interneurons in culture as well as in the intact spinal cord. We have combined extracellular multisite recording using multielectrode arrays with whole cell recordings to investigate the mechanisms involved in bursting in organotypic and dissociated cultures from the spinal cords of embryonic rats. Network bursts induced depolarization and spikes in single neurons, which were mediated by recurrent excitation through glutamatergic synaptic transmission. When such transmission was blocked, bursting ceased. However, tonic spiking persisted in some of the neurons. In such neurons intrinsic spiking was suppressed following the bursts and reappeared in the intervals after several seconds. The suppression of intrinsic spiking could be reproduced when, in the absence of fast synaptic transmission, bursts were mimicked by the injection of current pulses. Intrinsic spiking was also suppressed by a slight hyperpolarization. An afterhyperpolarization following the bursts was found in roughly half of the neurons. These afterhyperpolarizations were combined with a decrease in excitability. No evidence for the involvement of synaptic depletion or receptor desensitization in bursting was found, because neither the rate nor the size of spontaneous excitatory postsynaptic currents were decreased following the bursts. Extracellular stimuli paced bursts at low frequencies, but failed to induce bursts when applied too soon after the last burst. Altogether these results suggest that bursting in spinal cultures is mainly based on intrinsic spiking in some neurons, recurrent excitation of the network and auto-regulation of neuronal excitability.     

INaP underlies intrinsic spiking and rhythm generation in networks of cultured rat spinal cord neurons

We have shown previously that rhythm generation in disinhibited spinal networks is based on intrinsic spiking, network recruitment and a network refractory period following the bursts. This refractory period is based mainly on electrogenic Na/K pump activity. In the present work, we have investigated the role of the persistent sodium current (INaP) in the generation of bursting using patch-clamp and multielectrode array recordings. We detected INaP exclusively in the intrinsic spiking cells. The blockade of INaP by riluzole suppressed the bursting by silencing the intrinsic spiking cells and suppressing network recruitment. The blockade of the persistent sodium current produced a hyperpolarization of the membrane potential of the intrinsic spiking cells, but had no effect on non-spiking cells. We also investigated the involvement of the hyperpolarization-activated cationic current (I(h)) in the rhythmic activity. The bath application of ZD7288, a specific I(h) antagonist, slowed down the rate of the bursts by increasing the interburst intervals. I(h) was present in approximately 70% of the cells, both in the intrinsic spiking cells as well as in the non-spiking cells. We also found both kinds of cells in which I(h) was not detected. In summary, in disinhibited spinal cord cultures, a persistent sodium current underlies intrinsic spiking, which, via recurrent excitation, generates the bursting activity. The hyperpolarization-activated cationic current contributes to intrinsic spiking and modulates the burst frequency.     

The functional role of metabotropic glutamate receptors in epileptiform activity induced by 4-aminopyridine in the rat amygdala slice

The metabotropic glutamate receptor (mGluR) antagonist, (RS)-α-methyl-4-carboxyphenylglycine (MCPG; 500 μM), was tested on intracellularly recorded epileptiform activity induced by 4-aminopyridine (4-AP) in amygdala neurons. Superfusing 4-AP (1 mM) produced interictal spiking followed by ictal bursting. MCPG prevented the progressive transition from interictal spiking to ictal bursting but affected neither induction of interictal spiking nor maintenance of ongoing ictal bursting. These data suggest that mGluRs may be involved in the induction of ictal seizure events.     

Rhythmic intrinsic bursting neurons in human neocortex obtained from pediatric patients with epilepsy

Neocortical oscillations result from synchronized activity of a synaptically coupled network and can be strongly influenced by the intrinsic firing properties of individual neurons. As such, the intrinsic electroresponsive properties of individual neurons may have important implications for overall network function. Rhythmic intrinsic bursting (rIB) neurons are of particular interest, as they are poised to initiate and/or strongly influence network oscillations. Although neocortical rIB neurons have been recognized in multiple species, the current study is the first to identify and characterize rIB neurons in the human neocortex. Using whole-cell current-clamp recordings, rIB neurons (n = 12) are identified in human neocortical tissue resected from pediatric patients with intractable epilepsy. In contrast to human regular spiking neurons (n = 12), human rIB neurons exhibit rhythmic bursts of action potentials at frequencies of 0.1-4 Hz. These bursts persist after blockade of fast excitatory neurotransmission and voltage-gated calcium channels. However, bursting is eliminated by subsequent application of the persistent sodium current (I(NaP)) blocker, riluzole. In the presence of riluzole (either 10 or 20 μm), human rIB neurons no longer burst, but fire tonically like regular spiking neurons. These data demonstrate that I(NaP) plays a critical role in intrinsic oscillatory activity observed in rIB neurons in the human neocortex. It is hypothesized that aberrant changes in I(NaP) expression and/or function may ultimately contribute to neurological diseases that are linked to abnormal network activity, such as epilepsy.     

Fast-activating voltage- and calcium-dependent potassium (BK) conductance promotes bursting in pituitary cells: a dynamic clamp study

The electrical activity pattern of endocrine pituitary cells regulates their basal secretion level. Rat somatotrophs and lactotrophs exhibit spontaneous bursting and have high basal levels of hormone secretion, while gonadotrophs exhibit spontaneous spiking and have low basal hormone secretion. It has been proposed that the difference in electrical activity between bursting somatotrophs and spiking gonadotrophs is due to the presence of large conductance potassium (BK) channels on somatotrophs but not on gonadotrophs. This is one example where the role of an ion channel type may be clearly established. We demonstrate here that BK channels indeed promote bursting activity in pituitary cells. Blocking BK channels in bursting lacto-somatotroph GH4C1 cells changes their firing activity to spiking, while further adding an artificial BK conductance via dynamic clamp restores bursting. Importantly, this burst-promoting effect requires a relatively fast BK activation/deactivation, as predicted by computational models. We also show that adding a fast-activating BK conductance to spiking gonadotrophs converts the activity of these cells to bursting. Together, our results suggest that differences in BK channel expression may underlie the differences in electrical activity and basal hormone secretion levels among pituitary cell types and that the rapid rate of BK channel activation is key to its role in burst promotion.     

Antidromic activation of dorsal raphe neurons from neostriatum: Physiological characterization and effects of terminal autoreceptor activation

Three types of neurons, distinguished on the basis of their spontaneous firing rates and patterns, extracellularly recorded waveforms and responses to neostriatal stimulation, were observed in the dorsal raphe nucleus in urethane-anesthetized rats. Type 1 neurons (presumed to be serotonergic) fired spontaneously from 0.1 to 3 spikes/s in a regular pattern, with initial positive-going bi- or triphasic action potentials. Type 1 cells exhibited long-latency antidromic responses to neostriatal stimulation (mean +/- S.E.M. 24.9 +/- 0.3 ms) that sometimes occurred at discrete multiple latencies, and supernormal periods persisting up to 100 ms following spontaneous spikes. Type 2 cells fired spontaneously in an irregular, somewhat bursty pattern from 0 to 2 spikes/s with initial negative-going biphasic spikes, and were antidromically activated from neostriatal stimulation at shorter latencies than Type 1 cells (21.8 +/- 0.9 ms). Type 3 cells were characterized by initial positive-going biphasic waveforms and displayed a higher discharge rate (5-30 spikes/s) than Type 1 or Type 2 cells. Type 3 cells could not be antidromically activated from neostriatal stimulation. The relatively long conduction time to neostriatum of the Type 1 presumed serotonergic neuron is discussed with respect to previous interpretations of the synaptic action of serotonin in the neostriatum. In conjunction with these antidromic activation studies, the neurophysiological consequences of serotonergic terminal autoreceptor activation were examined by measuring changes in the excitability of serotonergic terminal fields in the neostriatum following administration of the serotonin autoreceptor agonist, 5-methoxy-N,N-dimethyltryptamine (5-MeODMT). The excitability of serotonergic terminal fields was decreased by intravenous injection of 40 micrograms/kg 5-MeODMT, and by infusion of 10-50 microM 5-MeODMT directly into the neostriatum. These results are interpreted from the perspective of mechanisms underlying autoreceptor-mediated regulation of serotonin release.     

Firing properties of frog tectal neurons in vitro

Whole-cell recordings from frog tectal slices revealed different types of neuronal firing patterns in response to prolonged current injection. The patterns included regular spiking without adaptation, accelerating firing, adapting spiking, repetitive bursting and phasic response with only one spike. The observed firing patterns are similar to those found in the mammalian superior colliculus. The frog tectum could be a useful preparation in elucidating the relationship between neuronal function and membrane properties.     

Multiple generators of 600 Hz wavelets in human SEP unmasked by varying stimulus rates.

Human scalp-derived somatosensory evoked potentials contain a high-frequency wavelet burst, presumably reflecting repetitive synchronized population spikes. Here, the burst refractory behavior was characterized using median nerve electrostimulation with 18 frequencies (0.5-25Hz) for comparison with cellular burst characteristics. Above 10 Hz only a brief high-frequency (700 Hz) burst component remained discernible, which gradually decreased; possible generators comprise cells capable of generating spike bursts of extraordinarily high frequency, such as pyramidal 'chattering cells', cortical fast spiking inhibitory interneurons and some thalamocortical relay cells. At stimulation frequencies <4 Hz an additional late burst component appeared with only 494 Hz intraburst frequency. Comparably long refractory periods and low intraburst frequencies have been described for bursting cells driven by low-threshold calcium currents.     

Metabolic and neural activity in the song system nucleus robustus archistriatalis: effect of age and gender

The sexually dimorphic robust archistriatal nucleus (RA) represents the telencephalic output of the bird song system. Here, we document sex-dependent changes in both the metabolic and neuronal activity of RA during the sensory and sensorimotor phases of song learning. From posthatching day (PHD) 20-63 in males but not females, RA and its input nucleus HVc showed sharp increases in cytochrome oxidase (CO) activity relative to surrounding archistriatum and the underlying shelf, respectively. In urethane-anesthetized birds, during the same period, the spontaneous activity of male RA neurons underwent dramatic changes in firing rate, distribution of interspike intervals, and bursting frequency, compared with other archistriatal cells. At PHD 20-21, RA neurons had extremely slow, irregular firing rates in birds of both sexes. In males, from PHD 30-36, RA neurons increased their firing rates and spiking activity became more regular, and at approximately PHD 38, strong bursts followed by inhibition (which in awake animals is associated with singing) began to be observed. Dual recordings from RA and HVc revealed synchronous bursting, with RA spikes lagging approximately 10 msec behind HVc. We conclude that changes in relative CO activity correlate with changes in spontaneous firing rates within RA and that patterns of RA spontaneous activity exhibit gradual change as birds enter early song and then again for plastic song. The emergence of strong burst patterns in RA occurs later in life than does input from HVc as established by tracer studies or based on observed HVc bursting in young animals.     

Noradrenaline does not change the mode of discharge of auditory cortex neurons

The mode of discharge of auditory cortex cells was studied during iontophoretic application of noradrenaline (NA). Only seven of 190 cells showed changes in interspike interval distribution during NA application. A similar conclusion was drawn when the analysis focused on 68 cells classified as bursting (n = 15), regular spiking (n = 49) or thin spike (n = 4) cells. Only two bursting cells showed changes in their ISI distribution. The effects on the mode of discharge were independent of the effect on the spike rate and were not a function of cortical depth. These results suggest that the changes in firing mode previously described in vitro occur for a limited percentage of cells and/or for cell types not very often recorded in vivo.     

Stochastic description of complex and simple spike firing in cerebellar Purkinje cells

Cerebellar Purkinje cells generate two distinct types of spikes, complex and simple spikes, both of which have conventionally been considered to be highly irregular, suggestive of certain types of stochastic processes as underlying mechanisms. Interestingly, however, the interspike interval structures of complex spikes have not been carefully studied so far. We showed in a previous study that simple spike trains are actually composed of regular patterns and single interspike intervals, a mixture that could not be explained by a simple rate-modulated Poisson process. In the present study, we systematically investigated the interspike interval structures of separated complex and simple spike trains recorded in anaesthetized rats, and derived an appropriate stochastic model. We found that: (i) complex spike trains do not exhibit any serial correlations, so they can effectively be generated by a renewal process, (ii) the distribution of intervals between complex spikes exhibits two narrow bands, possibly caused by two oscillatory bands (0.5-1 and 4-8 Hz) in the input to Purkinje cells and (iii) the regularity of regular patterns and single interspike intervals in simple spike trains can be represented by gamma processes of orders, which themselves are drawn from gamma distributions, suggesting that multiple sources modulate the regularity of simple spike trains.     

Intrinsic membrane properties and morphological characteristics of interneurons in the rat supratrigeminal region

The membrane properties and morphological features of interneurons in the supratrigeminal area (SupV) were studied in rat brain slices using whole-cell patch clamp recording techniques. We classified three morphological types of neurons as fusiform, pyramidal, and multipolar and four physiological types of neurons according to their discharge pattern in response to a 1-sec depolarizing current pulse from -80 mV. Single-spike neurons responded with a single spike, phasic neurons showed an initial burst of spikes and were silent during the remainder of the stimulus, delayed-firing (DF) neurons exhibited a slow depolarization and delay to initial spike onset, and tonic (T) neurons showed maintained a discharge throughout the stimulus pulse. In a subpopulation of neurons (10%), membrane depolarization to around -44 mV produced a rhythmic burst discharge (RB) that was associated with voltage-dependent subthreshold membrane oscillations. Both these phenomena were blocked by the sodium channel blocker riluzole at a concentration that did not affect the fast transient spike. Low doses of 4-AP, which blocks low-threshold K+ currents, transformed bursting into low-frequency tonic discharge. In contrast, bursting occurred with exposure to cadium, a calcium-channel blocker. This suggests that persistent sodium currents and low-threshold K+ currents have a role in intrinsic burst generation. Importantly, RB cells were most often associated with multipolar neurons that exhibited either a DF or a T discharge. Thus, the SupV contains a variety of physiological cell types with unique morphologies and discharge characteristics. Intrinsic bursting neurons form a unique group in this region. .     

Endogenously generated spontaneous spiking activities recorded from postnatal spiral ganglion neurons in vitro

Spontaneous spiking activities in the nervous system play an important role in the neuronal development and the coding of sensory information. Such firings could be initiated by transmitter leaked from the first-order sensory receptors or as a result of the internal operation of voltage-dependent ion channels intrinsic to the neuron. We recorded endogenously-generated spontaneous action potentials (APs) from postnatal spiral ganglion (SG) neurons of mouse in vitro. SG neurons in cultures displayed statistically stable spontaneous firings with no obvious bursting, rhythmic spiking and long silent gaps for as long as the recording configuration could be maintained. Average firing rates ranged from less than 1 to over 10 spikes/s, with most cells fired around 4 spikes/s. Interpulse interval histograms were remarkably similar to those recorded in vivo from the auditory nerve, with characteristics of a Poisson-like distribution. Resting membrane potential greatly altered the AP width and the rate of spontaneous firings. Spontaneous firing rates were also found to be controlled by the availability of the Shaw-like potassium channels. In contrast, matured SG neurons did not display any spontaneous APs, probably due to a large increase in the expression of the whole-cell potassium currents in comparison to their postnatal counterparts. This study provided the first direct evidence that postnatal SG neurons were capable of generating spontaneous APs independent of inputs from hair cells. Intracellular mechanisms for generating the spontaneous random spikes and the possible roles of such spontaneous activities in the postnatal development of SG neurons are discussed.