Cholinergic-mediated response enhancement in barrel cortex layer V pyramidal neurons

Neocortical cholinergic activity plays a fundamental role in sensory processing and cognitive functions, but the underlying cellular mechanisms are largely unknown. We analyzed the effects of acetylcholine (ACh) on synaptic transmission and cell excitability in rat "barrel cortex" layer V (L5) pyramidal neurons in vitro. ACh through nicotinic and M1 muscarinic receptors enhanced excitatory postsynaptic currents and through nicotinic and M2 muscarinic receptors reduced inhibitory postsynaptic currents. These effects increased excitability and contributed to the generation of Ca(2+) spikes and bursts of action potentials (APs) when inputs in basal dendrites were stimulated. Ca(2+) spikes were mediated by activation of NMDA receptors (NMDARs) and L-type voltage-gated Ca(2+) channels. Additionally, we demonstrate in vivo that basal forebrain stimulation induced an atropine-sensitive increase of L5 AP responses evoked by vibrissa deflection, an effect mainly due to the enhancement of an NMDAR component. Therefore, ACh modified the excitatory/inhibitory balance and switched L5 pyramidal neurons to a bursting mode that caused a potent and sustained response enhancement with possible fundamental consequences for the function of the barrel cortex.     

Cell size-dependent Nogo-A expression in layer V pyramidal neurons of the rat primary somatosensory cortex

Nogo-A mRNA and protein are present in the perikarya of neurons in both the intact and injured CNS. The present study focused primarily on Nogo-A protein expression in the primary somatosensory cortex of the adult rat. Coronal brain sections were probed with double-immunofluorescent labeling against Nogo-A together with NeuN, RBPC, or MAP-2 for confocal imaging. The sizes of the cell somata in pyramidal neurons and the thicknesses of neurites were measured on the captured confocal images. Nogo-A was expressed in larger pyramidal neurons and thicker neurites in layer V, but not in smaller pyramidal neurons and thinner neurites. Considering the morphological properties and the cell soma size reported in previous studies together with the present data, Nogo-A-positive neurons of layer V appear to be intrinsically bursting neurons that project axons to the subcortical regions. This suggests that intraneuronal Nogo-A may play roles in neurite growth and axonal regeneration of the corticofugal neurons, but not of columnar intrinsic neurons, in layer V of the S1 barrel cortex. Additionally, this study demonstrates a novel result, which is that layer V pyramidal neurons of the S1 barrel cortex exhibit a pattern of cell size-dependent intraneuronal Nogo-A expression.     

Maturation of layer V pyramidal neurons in the rat prefrontal cortex: intrinsic properties and synaptic function

Layer V pyramidal neurons in the rat medial prefrontal cortex (PFC) were examined with whole cell patch-clamp recording in acute slices from postnatal day 1 (P1) to P36. In the first few days after birth, layer V pyramidal neurons had low resting potentials, high-input resistance, and long membrane time constant. During the next 2 wk, the resting potential shifted by -14 mV, while the input resistance and time constant decreased by 15- and 4-fold, respectively. Between P3 and P21, the surface area of the cell body doubled, while the total lengths of apical and basal dendrites increased by 5- and 13-fold, respectively. Action potentials (APs) were observed at all aged tested. The peak amplitude of APs increased by 30 mV during the first 3 wk, while AP rise time and half-maximum duration shortened significantly. Compared with neurons at P21 or older, neurons in the first week required much smaller currents to reach their maximum firing frequencies, but the maximum frequencies were lower than those at older ages. Stimulation of layer II/III induced monosynaptic responses in neurons older than P5. Paired-pulse responses showed a short-term depression at P7, which shifted progressive to facilitation at older ages. These results demonstrate that, similar to other neurons in the brain, layer V pyramidal neurons in the PFC undergo a period of rapid development during the first 3 wk after birth. These findings suggest that the intrinsic properties of neurons and the properties of synaptic inputs develop concomitantly during early life.     

Orexin-A excites pyramidal neurons in layer 2/3 of the rat prefrontal cortex

The arousal peptides, orexins, play an important role in regulating the function of the prefrontal cortex (PFC). Although orexins have been shown to increase the excitability of deep-layer neurons in the medial prefrontal cortex (mPFC), little is known about their effect on layer 2/3, the main intracortical processing layer. In this study, we investigated the effect of orexin-A on pyramidal neurons in layer 2/3 of the mPFC using whole-cell recordings in rat brain slices. We observed that orexin-A reversibly depolarized layer 2/3 pyramidal neurons through a postsynaptic action. This depolarization was concentration-dependent and mediated via orexin receptor 1. In voltage-clamp recordings, the orexin-A-induced current was reduced by the replacement of internal K(+) with Cs(+), removal of external Na(+), or an application of flufenamic acid (an inhibitor of nonselective cation channels). A blocker of Na(+)/Ca(2+) exchangers (SN-6) did not influence the excitatory effect of orexin-A. Moreover, the current induced by orexin-A reversed near E(k) when the external solution contained low levels of Na(+). When recording with Cs(+)-containing pipettes in normal external solution, the reversal potential of the current was approximately -25 mV. These data suggest an involvement of both K(+) channels and nonselective cation channels in the effect of orexin-A. The direct excitatory action of orexin-A on layer 2/3 mPFC neurons may contribute to the modulation of PFC activity, and play a role in cognitive arousal.     

Multiple effects of dopamine on layer V pyramidal cell excitability in rat prefrontal cortex

The mechanisms underlying the inhibitory effects of dopamine (DA) on layer V pyramidal neuron excitability in the prelimbic region of the rat medial prefrontal cortex were investigated. Under control conditions, DA depressed both action potential generation (driven by somatic current injection) and input resistance (R(N)). The presence of GABA(A) receptor antagonists blocked DA-induced depression of action potential generation and revealed a delayed increase in excitability that persisted for the duration of experimental recording, up to 20 min following the washout of DA. In contrast to spike generation, disinhibition did not affect the transient depression of R(N) produced by DA, suggesting independent actions of DA on spike generation and R(N). Consistent with the hypothesis that DA acts to decrease pyramidal cell output via a GABAergic mechanism, DA increased the frequency of spontaneous inhibitory postsynaptic currents in both the absence and presence of TTX. Furthermore focal application of GABA to a perisomatic region mimicked the inhibitory effect of DA on spike production without affecting R(N). Focal application of bicuculline to the same location reversed the inhibitory effect of bath-applied DA on spike generation, while again having no effect on R(N). The depression of R(N) by DA was both occluded and mimicked by the Na(+) channel blocker TTX, suggesting the involvement of a Na(+) conductance in reducing pyramidal cell R(N) during the acute presence of DA. Together these data demonstrate that the acute presence of DA decreases pyramidal neuron excitability by two independent mechanisms. At the same time DA triggers a delayed and longer-lasting increase in excitability that is partially masked by synaptic inhibition.     

Desensitization of group I metabotropic glutamate receptors in rat sympathetic neurons

Desensitization of heterologously expressed metabotropic glutamate receptor 5a (mGluR5a) was examined in rat sympathetic neurons. Calcium currents in cells expressing mGluR5a exhibited substantial inhibition in response to glutamate exposure. In the continued presence of glutamate, inhibition attenuated rapidly over the course of about a minute. Desensitization was eliminated when a nonhydrolyzable ATP analogue was substituted for ATP in the pipette solution, suggesting that desensitization was mediated by a phosphorylation event. Next, pharmacological agents were used to investigate the nature of the kinase involved in desensitization. Desensitization was sensitive to the nonspecific kinase inhibitor, staurosporine, but not H-7, another nonspecific kinase inhibitor. Inhibitors of myosin light chain kinase and calmodulin-dependent kinase were without effect on desensitization. However, desensitization was sensitive to the protein kinase C inhibitor bisindolymaleimide. In contrast, G?6976, a selective inhibitor of conventional protein kinase C isoforms, was without effect. In addition, desensitization persisted in the presence of 10 mM intracellular bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, a fast Ca(2+) chelator. Finally, overexpression of wild-type calmodulin, which can bind mGluR5 and inhibit phosphorylation, did not alter mGluR desensitization. Two Ca(2+)-binding-deficient calmodulin mutants were also without effect. These data indicate a role for nonconventional protein kinase C isoforms as a mediator of mGluR5 desensitization and that the phosphorylation of mGluR5a that competes with calmodulin binding does not mediate desensitization.     

Distance- and activity-dependent modulation of spike back-propagation in layer V pyramidal neurons of the medial entorhinal cortex

Layer V principal neurons of the medial entorhinal cortex receive the main hippocampal output and relay processed information to the neocortex. Despite the fundamental role hypothesized for these neurons in memory replay and consolidation, their dendritic features are largely unknown. High-speed confocal and two-photon Ca(2+) imaging coupled with somatic whole cell patch-clamp recordings were used to investigate spike back-propagation in these neurons. The Ca(2+) transient associated with a single back-propagating action potential was considerably smaller at distal dendritic locations (>200 μm from the soma) compared with proximal ones. Perfusion of Ba(2+) (150 μM) or 4-aminopyridine (2 mM) to block A-type K(+) currents significantly increased the amplitude of the distal, but not proximal, Ca(2+) transients, which is strong evidence for an increased density of these channels at distal dendritic locations. In addition, the Ca(2+) transients decreased with each subsequent spike in a 20-Hz train; this activity-dependent decrease was also more prominent at more distal locations and was attenuated by the perfusion of the protein kinase C activator phorbol-di-acetate. These data are consistent with a phosphorylation-dependent control of back-propagation during trains of action potentials, attributable mainly to an increase in the time constant of recovery from voltage-dependent inactivation of dendritic Na(+) channels. In summary, dendritic Na(+) and A-type K(+) channels control spike back-propagation in layer V entorhinal neurons. Because the activity of these channels is highly modulated, the extent of the dendritic Ca(2+) influx is as well, with important functional implications for dendritic integration and associative synaptic plasticity.     

Activation of presynaptic group III metabotropic glutamate receptors depresses spontaneous inhibition in layer V of the rat entorhinal cortex

Whole cell voltage clamp recording was used to investigate neurotransmitter release onto neurones in deep and superficial layers of rat entorhinal cortex in vitro. Activation of metabotropic glutamate receptors with the agonist (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid depressed spontaneous release of the inhibitory neurotransmitter GABA in layer V, but not in layer II. Depression of transmitter release did not persist in the presence of the sodium channel blocker tetrodotoxin. It seems likely that activation of presynaptic glutamate heteroreceptors inhibits action potential dependent release of neurotransmitter via a direct action at the presynaptic terminal. We confirmed that depression of inhibitory neurotransmission in layer V was mediated by group III metabotropic glutamate receptors using a specific group III antagonist, (RS)-cyclopropyl-4-phosphonophenylglycine. Application of the antagonist alone did not alter the frequency of spontaneous neurotransmitter release, suggesting that the metabotropic glutamate receptor is not tonically active.In layer V of the entorhinal cortex, activation of presynaptic metabotropic glutamate receptors enhances spontaneous glutamate release, and inhibits spontaneous release of GABA. These effects may combine to increase random action potential firing in this layer, thereby reducing its capacity for synchrony generation. Our results are consistent with an anticonvulsant action for group III metabotropic glutamate receptors in the entorhinal cortex.     

Calcium currents in retrogradely labeled pyramidal cells from rat sensorimotor cortex

Our previous studies of calcium (Ca(2+)) currents in cortical pyramidal cells revealed that the percentage contribution of each Ca(2+) current type to the whole cell Ca(2+) current varies from cell to cell. The extent to which these currents are modulated by neurotransmitters is also variable. This study was directed at testing the hypothesis that a major source of this variability is recording from multiple populations of pyramidal cells. We used the whole cell patch-clamp technique to record from dissociated corticocortical, corticostriatal, and corticotectal projecting pyramidal cells. There were significant differences between the three pyramidal cell types in the mean percentage of L-, P-, and N-type Ca(2+) currents. For both N- and P-type currents, the range of percentages expressed was small for corticostriatal and corticotectal cells as compared with cells which project to the corpus callosum or to the general population. The variance was significantly different between cell types for N- and P-type currents. These results suggest that an important source of the variability in the proportions of Ca(2+) current types present in neocortical pyramidal neurons is recording from multiple populations of pyramidal cells.     

Properties of subthreshold response and action potential recorded in layer V neurons from cat sensorimotor cortex in vitro

Properties of the action potential and subthreshold response were studied in large layer V neurons in in vitro slices of cat sensorimotor cortex using intracellular recording and stimulation, application of agents that block active conductances, and a single-microelectrode voltage clamp (SEVC). A variety of measured parameters, including action-potential duration, afterpotentials, input resistance, rheobase, and membrane time constant, were similar to the same parameters reported for large neurons from this region of cortex in vivo. Action-potential amplitudes and resting potentials were greater in vitro. Most measured parameters were distributed unimodally, suggesting that these parameters are similar in all large layer V neurons irrespective of their axonal termination. The voltage response to subthreshold constant-current pulses exhibited both time and voltage dependence in the great majority of cells. Current pulses in either the hyperpolarizing or subthreshold depolarizing direction cause the membrane potential to attain an early peak and then decay (sag) to a steady level. On termination of the pulse, the membrane response transiently overshoots resting potential. Plots of current-voltage relations demonstrate inward rectification during polarization on either side of resting potential. Subthreshold inward rectification in the depolarizing direction is abolished by tetrodotoxin (TTX). The ionic currents responsible for subthreshold rectification and sag were examined using the SEVC. Steady inward rectification in the depolarizing direction is caused by a persistent, subthreshold sodium current (INaP) (54). Sag observed in response to a depolarizing current pulse is due to activation of a slow outward current, which superimposes on and partially counters the persistent sodium current. Both sag in response to hyperpolarizing current pulses and rectification in the hyperpolarizing direction are caused by a slow inward "sag current" that is activated by hyperpolarizing voltage steps. The sag current is unaltered by TTX, tetraethylammonium, (TEA), Co2+, Ba2+, or 4-aminopyridine. Fast-rising, short-duration action potentials can be elicited by an intracellular current pulse or by orthodromic or antidromic stimulation. Spikes are blocked by TTX. The form of the afterpotential following a directly evoked spike varies among cells with similar resting potentials. Biphasic afterhyperpolarizations (AHPs) with fast and slow components were most frequently seen. About 30% of the cells displayed a depolarizing afterpotential (DAP), which was often followed by an AHP. Other cells displayed a purely monophasic AHP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Different roles of group I and group II metabotropic glutamate receptors on phencyclidine-induced dopamine release in the rat prefrontal cortex

The dopamine system in the limbic-prefrontal cortex has been assumed to play an important role in the cognitive dysfunction of schizophrenia and phencyclidine (PCP)-induced psychosis. In the present study, the role of metabotropic glutamate (mGlu) receptor subtypes on PCP-induced cortical dopamine release was investigated using the microdialysis technique. Infusion of 50 and 100 microM of non-selective mGlu receptor agonist trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid inhibited PCP-induced dopamine release, while the basal dopamine level was not significantly affected. A similar inhibition of PCP-induced dopamine release was observed with 100 and 500 microM of selective group I mGlu receptor agonist, (+)-3-hydroxy-phenylglycine. On the other hand, infusion of 10 microM of selective group II mGlu receptor agonist, 2-(2, 3-dicarboxycyclopropyl)-glycine, enhanced the PCP-induced dopamine increase. These results suggest that group I and II mGlu receptors exert opposite modulations on the PCP-induced dopamine release.     

Properties of persistent sodium conductance and calcium conductance of layer V neurons from cat sensorimotor cortex in vitro

Properties of the persistent sodium conductance and the calcium conductance of layer V neurons from cat sensorimotor cortex were examined in an in vitro slice preparation by use of a single microelectrode, somatic voltage clamp, current clamp, intra- and extracellular application of blocking agents, and extracellular ion substitution. The persistent sodium current (INaP) attained its steady level within 2-4 ms of a step change in voltage at every potential where it could be examined directly [to about 40 mV positive to resting potential (RP)]. Because of its fast onset INaP can be activated during a single excitatory postsynaptic potential (EPSP) and can influence the subsequent voltage time course and cell excitability. Application of a depolarizing holding potential greater than or equal to 20 mV positive to RP could inactivate spikes, thus allowing examination of INaP at voltages positive to spike threshold. At every potential where INaP was visible, it was mixed with a slow outward current. After depressing potassium currents with blocking agents, INaP could be observed during depolarizations to about 40 mV positive to RP where it is normally hidden by the larger outward currents. Indirect evidence suggests that INaP is present and large during prolonged depolarizations greater than 50 mV positive to RP. INaP was blocked by intracellular injection of the lidocaine derivative QX-314, as well as by extracellular tetrodotoxin (TTX). INaP was much more sensitive to QX-314 than was the height and rate of rise of the spike. This observation and the results in paragraph 3 above are best explained by separate INaP and spike sodium channels. After blockade of INaP and sodium spikes, Ca2+ spikes could be evoked only if potassium currents were first depressed. The Ca2+-dependent nature of the regenerative potentials was indicated by their disappearance when Co2+ or Mn2+ was substituted for Ca2+ in the perfusate and by the appearance of greatly enhanced potentials of similar form when Ba2+ was substituted for Ca2+. Ba2+ substitution greatly enhanced evoked and spontaneous synaptic potentials. Prolonged-plateau action potentials could be evoked in the presence of TTX and Ba2+. Ca2+ spike threshold was 30-40 mV positive to RP, which is significantly more positive than sodium spike threshold. Results of voltage clamp in the normal perfusate and in the presence of Ca2+-blockers or Ba2+ indicated that little or no Ca2+ conductance is activated in the voltage range 25 mV positive to RP where INaP is the dominant ionic current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Net dendritic stability of layer II pyramidal neurons in F344 rat entorhinal cortex from 12 to 37 months.

Dendritic extent of Golgi-Cox stained layer II entorhinal cortex pyramidal neurons was quantified in five groups of male F344 rats aged 12, 20, 27, 30 and 37 months. Over the age range studied, neither the apical nor the basal dendritic trees showed any statistically significant change in total dendritic length, numbers of segments or average segment length. This finding of average stability of the dendritic tree does not imply absence of remodelling of connections, but does require that if remodeling does occur, retraction and proliferation of dendrites must, on average, be equal. We hypothesized that in groups of animals with similar genetic and environmental histories neighbor neuron death provides the major stimulus for dendritic proliferation. Since we found dendritic stability in the cells reported here, we would predict that there should be no age-related loss of layer II pyramidal neurons in the entorhinal cortex of the normally aging F344 male rat between 12 and 37 months. This hypothesis may be tested by counting neurons within this region.     

Voltage-gated sodium channels shape subthreshold EPSPs in layer 5 pyramidal neurons from rat prefrontal cortex

The role of voltage-dependent channels in shaping subthreshold excitatory postsynaptic potentials (EPSPs) in neocortical layer 5 pyramidal neurons from rat medial prefrontal cortex (PFC) was investigated using patch-clamp recordings from visually identified neurons in brain slices. Small-amplitude EPSPs evoked by stimulation of superficial layers were not affected by the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonopentanoic acid but were abolished by the AMPA receptor antagonist 6-cyano-7-nitroquinoxalene-2,3-dione, suggesting that they were primarily mediated by AMPA receptors. AMPA receptor-mediated EPSPs (AMPA-EPSPs) evoked in the apical dendrites were markedly enhanced, or increased in peak and duration, at depolarized holding potentials. Enhancement of AMPA-EPSPs was reduced by loading the cells with lidocaine N-ethylbromide (QX-314) and by local application of the Na(+) channel blocker tetrodotoxin (TTX) to the soma but not to the middle/proximal apical dendrite. In contrast, blockade of Ca(2+) channels by co-application of Cd(2+) and Ni(2+) to the soma or apical dendrite did not affect the AMPA-EPSPs. Like single EPSPs, EPSP trains were shaped by Na(+) but not Ca(2+) channels. EPSPs simulated by injecting synaptic-like current into proximal/middle apical dendrite (simEPSPs) were enhanced at depolarized holding potentials similarly to AMPA-EPSPs. Extensive blockade of Ca(2+) channels by bath application of the Cd(2+) and Ni(2+) mixture had no effects on simEPSPs, whereas bath-applied TTX removed the depolarization-dependent EPSP amplification. Inhibition of K(+) currents by 4-aminopyridine (4-AP) and TEA increased the TTX-sensitive EPSP amplification. Moreover, strong inhibition of K(+) currents by high concentrations of 4-AP and TEA revealed a contribution of Ca(2+) channels to EPSPs that, however, seemed to be dependent on Na(+) channel activation. Our results indicate that in layer 5 pyramidal neurons from PFC, Na(+), and K(+) voltage-gated channels shape EPSPs within the voltage range that is subthreshold for somatic action potentials.     

Populations of GABAergic neurons and axons in layer I of rat auditory cortex

Neurons and axon terminals (puncta) immunostained by an antibody against glutamic acid decarboxylase were studied in layer I of adult rats in architectonically identified area 41 of auditory cortex. The borders of area 41 and the laminar subdivisions of cortex were established in normal material and in other studies of cortical connections. Vibratomed or frozen sections were immunostained. The objectives of the study were to classify the types of (i) glutamic acid decarboxylase-positive neurons and (ii) puncta, and (iii) to examine their spatial distribution within layer I. Control sections were devoid of specific immunostaining. More than 90% of layer I cells are glutamic acid decarboxylase-positive. Four types of neuron were identified in Golgi material, including small neurons with stellate dendritic fields, horizontal cells with laterally projecting arbors, medium-sized neurons with stellate, widely ramifying dendritic fields, and large neurons with broad dendritic fields spanning the depth of layer I or branching laterally. In the glutamic acid decarboxylase material, examples with a somatodendritic shape matching each of these types were found. The average somatic diameter of glutamic acid decarboxylase-positive neurons (mean = 59 microns2, S.D. = 21 microns2) suggests that the small and medium-sized neurons predominate. Glutamic acid decarboxylase-positive neurons occur throughout the depth of layer I, but are far more numerous in the deeper half (68% in layer Ib) than in the superficial part (32% in layer Ia). Glutamic acid decarboxylase-positive neurons form small clusters of three to five cells across the cortical surface, with a range of 0-9/100 microns across the cortex. Most glutamic acid decarboxylase-positive neuronal perikarya were intensely immunostained, and the dendrites of the medium-sized and large neurons were traced as far as 50-75 microns beyond their initial branching point. Glutamic acid decarboxylase-positive puncta also had variable shapes. Both small, fine puncta (less than 0.5 micron in diameter) and larger, coarser ones (greater than 1.5 micron in diameter) were present, though the former were much more common. In traverses from the pia to the layer II border, the puncta average about 40/100 microns2 (range: 20-80), and the shape of individual pia--layer II traverses is multipeaked, often with a slight trough at congruent to 80 microns depth, then rising slowly in number toward layer II.(ABSTRACT TRUNCATED AT 400 WORDS)