Properties of mEPSCs recorded in layer II neurones of rat barrel cortex

Voltage-clamp recordings from layer II neurones in somatosensory cortex of rats aged between 12 and 17 days showed a high frequency of spontaneous postsynaptic currents (sPSCs), which on average was 33 ± 13 Hz ( s.d .). sPSCs were mediated largely by glutamatergic AMPA receptors. Their rates and amplitudes were independent of blocking sodium channels with 1 μ m tetrodotoxin (TTX). Most of them, therefore, represent genuine miniature excitatory postsynaptic currents (mEPSCs). The rise time of the fastest (10 %) mEPSCs was 288 ± 86 μs (10-90 %) and the half-width was 1073 ± 532 μs. The amplitude was −5.9 ± 1.1 pA with a coefficient of variation (CV) of 0.44 ± 0.14. The rate of mEPSCs was very temperature sensitive with a Q ₁₀ (33-37 °C) of 8.9 ± 0.9. Due to this temperature sensitivity, we estimated that the microscope lamp contributed an increase in temperature of about 4 °C to the tissue in the focal volume of the condenser. Cell-type differences in the rate of mEPSCs were found between pyramidal/multipolar and bipolar cells. The latter had a frequency of about a third of that seen in the other cell groups. Recordings in layer II are ideally suited to investigate mechanisms of spontaneous transmitter release.     

The contribution of intracellular calcium stores to mEPSCs recorded in layer II neurones of rat barrel cortex

Loading slices of rat barrel cortex with 50 μ m BAPTA-AM while recording from pyramidal cells in layer II induces a marked reduction in both the frequency and amplitudes of mEPSCs. These changes are due to a presynaptic action. Blocking the refilling of Ca²⁺ stores with 20 μ m cyclopiazonic acid (CPA), a SERCA pump inhibitor, in conjunction with neuronal depolarisation to activate Ca²⁺ stores, results in a similar reduction of mEPSCs to that observed with BAPTA-AM, indicating that the source for intracellular Ca²⁺ is the endoplasmic reticulum. Block or activation of ryanodine receptors by 20 μ m ryanodine or 10 m m caffeine, respectively, shows that a significant proportion of mEPSCs are caused by Ca²⁺ release from ryanodine stores. Blocking IP₃ receptors with 14 μ m 2-aminoethoxydiphenylborane (2APB) also reduces the frequency and amplitude of mEPSCs, indicating the involvement of IP₃ stores in the generation of mEPSCs. Activation of group I metabotropic receptors with 20 μ m ( RS) -3,5-dihydroxyphenylglycine (DHPG) results in a significant increase in the frequency of mEPSCs, further supporting the role of IP₃ receptors and indicating a role of group I metabotropic receptors in causing transmitter release. Statistical evidence is presented for Ca²⁺-induced Ca²⁺ release (CICR) from ryanodine stores after the spontaneous opening of IP₃ stores.     

Sub- and suprathreshold receptive field properties of pyramidal neurones in layers 5A and 5B of rat somatosensory barrel cortex

Layer 5 (L5) pyramidal neurones constitute a major sub- and intracortical output of the somatosensory cortex. This layer 5 is segregated into layers 5A and 5B which receive and distribute relatively independent afferent and efferent pathways. We performed in vivo whole-cell recordings from L5 neurones of the somatosensory (barrel) cortex of urethane-anaesthetized rats (aged 27–31 days). By delivering 6 deg single whisker deflections, whisker pad receptive fields were mapped for 16 L5A and 11 L5B neurones located below the layer 4 whisker-barrels. Average resting membrane potentials were −75.6 ± 1.1 mV, and spontaneous action potential (AP) rates were 0.54 ± 0.14 APs s⁻¹. Principal whisker (PW) evoked responses were similar in L5A and L5B neurones, with an average 5.0 ± 0.6 mV postsynaptic potential (PSP) and 0.12 ± 0.03 APs per stimulus. The layer 5A sub- and suprathreshold receptive fields (RFs) were more confined to the principle whisker than those of layer 5B. The basal dendritic arbors of layer 5A and 5B cells were located below both layer 4 barrels and septa, and the cell bodies were biased towards the barrel walls. Responses in both L5A and L5B developed slowly, with onset latencies of 10.1 ± 0.5 ms and peak latencies of 33.9 ± 3.3 ms. Contralateral multi-whisker stimulation evoked PSPs similar in amplitude to those of PW deflections; whereas, ipsilateral stimulation evoked smaller and longer latency PSPs. We conclude that in L5 a whisker deflection is represented in two ways: focally by L5A pyramids and more diffusely by L5B pyramids as a result of combining different inputs from lemniscal and paralemniscal pathways. The relevant output evoked by a whisker deflection could be the ensemble activity in the anatomically defined cortical modules associated with a single or a few barrel-columns.     

Excitation and depression of cortical neurones by 5-hydroxytryptamine

1. 5-Hydroxytryptamine (5-HT) and various 5-HT antagonists have been applied micro-electrophoretically from multibarrelled micropipettes into the environment of single neurones in the post-sigmoid and suprasylvian gyri of the cat cerebral cortex.2. In unanaesthetized animals (encéphale isolé) a high proportion of neurones (30%) were excited by 5-HT. This excitation usually had a rapid onset and was seen both in spontaneously active neurones and in otherwise quiescent neurones in which firing was induced by L-glutamate. Some neurones were so sensitive that the uncontrolled diffusion from micropipettes was sufficient to excite them. More cells were excited by 5-HT applied as a cation from solutions of the bimaleate salt than when solutions of the creatinine sulphate salt were used.3. In a high proportion of cells (33%) spontaneous firing or amino acid excitation was depressed by 5-HT.4. A mixed effect was seen in a small proportion (6%) of the cells tested; usually 5-HT caused an excitation initially which was followed by a depression. In other cells, desensitization occurred, and the excitatory effect of 5-HT was diminished or lost.5. When glutamate was used to excite otherwise quiescent cells, there was a significant increase in the number of cells excited by 5-HT and a significant decrease in the number of cells unaffected compared with spontaneously active cells.6. The micro-electrophoretic application of D-lysergic diethylamide (LSD 25), 2-brom LSD (BOL 148), methysergide (UML 491), or 2'- (3-dimethylaminopropylthio)cinnamanilide (SQ 10643) temporarily prevented excitation by 5-HT in half the cells tested. LSD and SQ 10643 were particularly potent in this respect. This antagonism of 5-HT excitation could still be seen when excitation of the cell by L-glutamate or acetylcholine (ACh) was unaffected.7. The depression induced by 5-HT was not prevented by the application of known 5-HT antagonists in the majority of the cells tested (93%). In two cells, however, the depression was reversibly prevented by these antagonists.8. Some cells tested with 5-HT were also tested with ACh or (-)-noradrenaline. The response of a cell to ACh was not significantly related to its response to 5-HT. The degree of correlation between the responses to noradrenaline and 5-HT was large, but not statistically significant with the small number of cells studied.9. The effects of 5-HT on cells in animals anaesthetized with alpha-chloralose did not differ significantly from its effects in unanaesthetized preparations. It is suggested that the use of this anaesthetic may prove a useful alternative to unanaesthetized preparations.10. The systemic injection of small quantities of thiopentone sodium selectively and reversibly reduced the sensitivity of some units to excitation by 5-HT at a time when the response to glutamate was unaffected. On other occasions, the 5-HT excitation was unaffected, though the response to glutamate was reduced.11. These results are discussed in relation to the possible nature of the 5-HT receptors in the cerebral cortex, and the interfering effects of anaesthesia on the response of brain cells to potential transmitter substances.     

Short-term dynamics of synaptic transmission within the excitatory neuronal network of rat layer 4 barrel cortex

The short-term plasticity of synaptic transmission between excitatory neurons within a barrel of layer 4 rat somatosensory neocortex was investigated. Action potentials in presynaptic neurons at frequencies ranging from 1 to 100 Hz evoked depressing postsynaptic excitatory postsynaptic potentials (EPSPs). Recovery from synaptic depression followed an exponential time course with best-fit parameters that differed greatly between individual synaptic connections. The average maximal short-term depression was close to 0.5 with a recovery time constant of around 500 ms. Analysis of each individual sweep showed that there was a correlation between the amplitude of the response to the first and second action potentials such that large first EPSPs were followed by smaller than average second EPSPs and vice versa. Short-term depression between excitatory layer 4 neurons can thus be termed use dependent. A simple model describing use-dependent short-term plasticity was able to closely simulate the experimentally observed dynamic behavior of these synapses for regular spike trains. More complex irregular trains of 10 action potentials occurring within 500 ms were initially well described, but during the train errors increased. Thus for short periods of time the dynamic behavior of these synapses can be predicted accurately. In conjunction with data describing the connectivity, this forms a first step toward computational modeling of the excitatory neuronal network of layer 4 barrel cortex. Simulation of whisking-evoked activity suggests that short-term depression may provide a mechanism for enhancing the detection of objects within the whisker space.     

Loss of sensory input increases the intrinsic excitability of layer 5 pyramidal neurons in rat barrel cortex

Development of the cortical map is experience dependent, with different critical periods in different cortical layers. Previous work in rodent barrel cortex indicates that sensory deprivation leads to changes in synaptic transmission and plasticity in layer 2/3 and 4. Here, we studied the impact of sensory deprivation on the intrinsic properties of layer 5 pyramidal neurons located in rat barrel cortex using simultaneous somatic and dendritic recording. Sensory deprivation was achieved by clipping all the whiskers on one side of the snout. Loss of sensory input did not change somatic active and resting membrane properties, and did not influence dendritic action potential (AP) backpropagation. In contrast, sensory deprivation led to an increase in the percentage of layer 5 pyramidal neurons showing burst firing. This was associated with a reduction in the threshold for generation of dendritic calcium spikes during high-frequency AP trains. Cell-attached recordings were used to assess changes in the properties and expression of dendritic HCN channels. These experiments indicated that sensory deprivation caused a decrease in HCN channel density in distal regions of the apical dendrite. To assess the contribution of HCN down-regulation on the observed increase in dendritic excitability following sensory deprivation, we investigated the impact of blocking HCN channels. Block of HCN channels removed differences in dendritic calcium electrogenesis between control and deprived neurons. In conclusion, these observations indicate that sensory loss leads to increased dendritic excitability of cortical layer 5 pyramidal neurons. Furthermore, they suggest that increased dendritic calcium electrogenesis following sensory deprivation is mediated in part via down-regulation of dendritic HCN channels.     

Angular tuning and velocity sensitivity in different neuron classes within layer 4 of rat barrel cortex

Local circuitry within layer IV whisker-related barrels is preferentially sensitive to thalamic population firing synchrony, and neurons respond most vigorously to stimuli, such as high-velocity whisker deflections, that evoke it. Field potential recordings suggest that thalamic barreloid neurons having similar angular preferences fire synchronously. To examine whether angular tuning of cortical neurons might also be affected by thalamic firing synchrony, we characterized responses of layer IV units to whisker deflections that varied in angular direction and velocity. Barrel regular-spike units (RSUs) became more tuned for deflection angle with slower whisker movements. Deflection amplitude had no affect. Barrel fast-spike units (FSUs) were poorly tuned for deflection angle, and their responses remained constant with different deflection velocity. The dependence of angular tuning on deflection velocity among barrel RSUs appears to reflect the same underlying response dynamics that determine their velocity sensitivity and receptive field focus. Unexpectedly, septal RSUs and FSUs are largely similar to their barrel counterparts despite available evidence suggesting that they receive different afferent inputs and are embedded within different local circuits.     

The dependence of response amplitude and variance of cat visual cortical neurones on stimulus contrast

Summary For neurones in the cat's striate cortex, we examined the dependence of response on the contrast of moving sinusoidal gratings. Most neurones showed a clear threshold contrast below which no response was elicited. Such thresholds presumably contribute to the animal's behavioural threshold, which should not be accounted for solely in terms of the detection of a signal in the presence of spontaneous noise. Above threshold, the response amplitude usually increased linearly with contrast until it began to saturate at the highest contrasts. The variance of the response increased with its amplitude; this finding perhaps underlies the Weber-Fechner relation for psychophysical contrast discrimination.     

Multiple forms of activity-dependent intrinsic plasticity in layer V cortical neurones in vivo

Synaptic plasticity is classically considered as the neuronal substrate for learning and memory. However, activity-dependent changes in neuronal intrinsic excitability have been reported in several learning-related brain regions, suggesting that intrinsic plasticity could also participate to information storage. Compared to synaptic plasticity, there has been little exploration of the properties of induction and expression of intrinsic plasticity in an intact brain. Here, by the means of in vivo intracellular recordings in the rat we have examined how the intrinsic excitability of layer V motor cortex pyramidal neurones is altered following brief periods of repeated firing. Changes in membrane excitability were assessed by modifications in the discharge frequency versus injected current ( F–I ) curves. Most (∼64%) conditioned neurones exhibited a long-lasting intrinsic plasticity, which was expressed either by selective changes in the current threshold or in the slope of the F–I curve, or by concomitant changes in both parameters. These modifications in the neuronal input–output relationship led to a global increase or decrease in intrinsic excitability. Passive electrical membrane properties were unaffected by the intracellular conditioning, indicating that intrinsic plasticity resulted from modifications of voltage-gated ion channels. These results demonstrate that neocortical pyramidal neurones can express in vivo a bidirectional use-dependent intrinsic plasticity, modifying their sensitivity to weak inputs and/or the gain of their input–output function. These multiple forms of experience-dependent intrinsic changes, which expand the computational abilities of individual neurones, could shape new network dynamics and thus might participate in the formation of mnemonic motor engrams.     

The cortical plate and molecular layer of the late rat fetus

Summary In the cortical plate of the late prenatal rat fetus the neuroblasts can be considered to be of three types: mature neuroblasts which are prominent in the lower levels of the cortical plate and have some of the cytoplasmic and nuclear features of neurons, immature neuroblasts that have recently completed their migrations into the cortical plate, and migrating neuroblasts that are still in the process of moving to their definitive positions. Both of these latter types have darker cytoplasm than the mature neuroblasts. All of the neuroblasts have an apical process that extends directly towards the pial surface of the cortical plate and a basal process that is directed towards the intermediate zone of the developing hemisphere. In Golgi preparations some of these basal processes, particularly those of neuroblasts situated in the lower levels of the cortical plate, seem to have formed axons that pass through the intermediate zone to enter the developing white matter, in which they turn at right angles away from, and rarely toward, the midline. Other elements traversing the cortical plate are the ascending processes of spongioblasts that branch in the molecular layer and form expansions at the surface of the hemisphere. In the molecular layer the spongioblast terminal branches intertwine with the apical tufts of the ascending neuroblast processes and with thin processes that have the features of axons, to form a loose neuropil. In the cortical plate the spongioblast processes are usually closely and preferentially surrounded by the dark migrating neuroblasts and by the immature neuroblasts. Both of these latter may partially encompass spongioblast processes. Hence it is concluded that the spongioblast processes act as guides along which the migrating neuroblasts ascend through the cortical plate.Supported by United States Public Health Service Research Grant NB 07016, from the National Institute of Neurological Diseases and Stroke.     

Local circuits targeting parvalbumin-containing interneurons in layer IV of rat barrel cortex

Interactions between inhibitory interneurons and excitatory spiny neurons and also other inhibitory cells represent fundamental network properties which cause the so-called thalamo-cortical response transformation and account for the well-known receptive field differences of cortical layer IV versus thalamic neurons. We investigated the currently largely unknown morphological basis of these interactions utilizing acute slice preparations of barrel cortex in P19-21 rats. Layer IV spiny (spiny stellate, star pyramidal and pyramidal) neurons or inhibitory (basket and bitufted) interneurons were electrophysiologically characterized and intracellularly biocytin-labeled. In the same slice, we stained parvalbumin-immunoreactive (PV-ir) interneurons as putative target cells after which the tissue was subjected to confocal image acquisition. Parallel experiments confirmed the existence of synaptic contacts in these types of connection by correlated light and electron microscopy. The axons of the filled neurons differentially targeted barrel PV-ir interneurons: (1) The relative number of all contacted PV-ir cells within the axonal sphere was 5–17% for spiny (n = 10), 32 and 58% for basket (n = 2) and 12 and 13% for bitufted (n = 2) cells. (2) The preferential subcellular site which was contacted on PV-ir target cells was somatic for four and dendritic for five spiny cells; for basket cells, there was a somatic and for bitufted cells a dendritic preference in each examined case. (3) The highest number of contacts on a single PV-ir cell was 9 (4 somatic and 5 dendritic) for spiny neurons, 15 (10 somatic and 5 dendritic) for basket cells and 4 (1 somatic and 3 dendritic) for bitufted cells. These patterns suggest a cell type-dependent communication within layer IV microcircuits in which PV-ir interneurons provide not only feed-forward but also feedback inhibition thus triggering the thalamo-cortical response transformation.     

Phosphonate analogues of carboxylic acids as aminoacid antagonists on rat cortical neurones

Phosphonate analogues of carboxylic acids have been tested as antagonists of excitatory aminoacids in rat cerebral cortex. (+/-)--2-Amino-7-phosphono-heptanoic acid and the (-)-D-isomer of the pentanoate derivative were more potent and selective antagonists of N-methyl-D-aspartate (NMDA) than compounds tested previously. The results support the view that a distinct population of receptors exists which are preferentially activated by NMDA.     

Inter- and intralaminar subcircuits of excitatory and inhibitory neurons in layer 6a of the rat barrel cortex

Approximately half the excitatory neurons in layer 6 (L6) of the rat barrel cortex project to the thalamus with axon collaterals ramifying in the granular L4; the remaining project within cortex with collaterals restricted to infragranular laminae. In analogy, L6 inhibitory neurons also include locally arborizing and inter-laminar projecting neurons. We examined whether L6 neurons participating in different laminar interactions were also morphologically and electrically distinct. Corticothalamic (CT) neurons were labeled by in vivo injections of a retrogradely transported fluorescent tracer into the primary thalamic nucleus. Whole cell current-clamp recordings were performed from labeled and unlabeled L6 neurons in brain slices of juvenile rats; the morphology of cells was subsequently recovered and reconstructed. Corticocortical (CC) neurons were distinguished from CT cells based on the absence of a subcortical projection and the predominantly infragranular arborization of their axon collaterals. Two morphological CC subtypes could be further distinguished based on the structure of their apical dendrite. Electrically, CT neurons had shorter membrane time-constants and action potential (AP) durations and higher rheobase currents. CC neurons fired high-frequency spike doublets or triplets on sustained depolarization; the burst frequency also distinguished the two morphological CC subtypes. Among inhibitory L6 cells, the L4-projecting (L6i_L4) and local (L6i_L6) inhibitory neurons also had contrasting firing properties; L6i_L4 neurons had broader APs and lower maximal firing rates. We propose that L6 excitatory and inhibitory neurons projecting to L4 constitute specialized subcircuits distinct from the infragranular network in their connectivity and firing patterns.     

Electrical stimulation of locus coeruleus strengthens the surround inhibition in layer V barrel cortex in rat

It is believed that locus coeruleus (LC) influences the sensory information processing. However, its role in cortical surround inhibitory mechanism is not well established. In this experiment, using controlled mechanical displacement of whiskers; we investigated the effect of electrical stimulation of LC on response of layer V barrel cortical neurons in anesthetized rat. LC was stimulated 0, 50, 100, 200 and 400 ms before principal or adjacent whiskers deflection. For assessing the effect of LC stimulation on inhibitory receptive filed of barrel neurons, adjacent whisker was also deflected 20 ms before principal whisker deflection, and LC stimulation was applied 0-400 ms before principal whisker displacement. We found that LC stimulation increase the response magnitude of layer V neurons to principal whisker deflection (significant in 50-400 ms intervals). This increase was also observed in response to adjacent whisker deflection (significant in 100 ms interval). The response latency of neurons was decreased when LC was stimulated 400 ms before principal whisker deflection but LC stimulation did not affect the neuronal response latency to adjacent whisker displacement. Inhibitory effect of adjacent whisker deflection on neuronal response magnitude was increased by LC stimulation, tested in combined whisker displacement. These findings suggest that LC, by modulating the neuronal responses, enhances the neuronal responsiveness to sensory stimuli and increases their surround inhibition in cortex.