Tadaharu Tsumoto

GABAergic neurons are less selective to stimulus orientation than excitatory neurons in layer II/III of visual cortex, as revealed by in vivo functional Ca2+ imaging in transgenic mice.

Most neurons in the visual cortex are selectively responsive to visual stimulation of a narrow range of orientations, and GABAergic neurons are considered to play a role in the formation of such orientation selectivity. This suggests that response properties of GABAergic neurons may be different from those of excitatory neurons. This view remains unproved, however. To address this issue, we applied in vivo two-photon functional Ca2+ imaging to transgenic mice, in which GABAergic neurons express enhanced green fluorescent protein. Astroglia were stained by an astrocyte-specific dye. The three types of cells, GABAergic neurons, excitatory neurons, and astrocytes, in layer II/III of the visual cortex were differentially identified by using different wavelengths of excitation light and a dichroic mirror for emitted fluorescence, and their responses to moving visual stimuli at different orientations were measured with changes in the intensity of fluorescence of a Ca2+-sensitive dye. We found that almost all GABAergic neurons have orientation-insensitive responses, whereas most of excitatory neurons have orientation-selective responses.

Change of conduction velocity by regional myelination yields constant latency irrespective of distance between thalamus and cortex

The widely spanning sensory cortex receives inputs from the disproportionately smaller nucleus of the thalamus, which results in a wide variety of travelling distance among thalamic afferents. Yet, latency from the thalamus to a cortical cell is remarkably constant across the cortex (typically, ≈2 ms). Here, we found a mechanism that produces invariability of latency among thalamocortical afferents, irrespective of the variability of travelling distances. The conduction velocity (CV) was calculated from excitatory postsynaptic currents recorded from layer IV cells in mouse thalamocortical slices by stimulating the ventrobasal nucleus of the thalamus (VB) and white matter (WM). In adults, the obtained CV for VB to WM (CVVB-WM; 3.28 ± 0.11 m/s) was ≈10 times faster than that of WM to layer IV cells (CVWM-IV; 0.33 ± 0.05 m/s). The CVVB-WM was confirmed by recording antidromic single-unit responses from VB cells by stimulating WM. Exclusion of synaptic delay from CVWM-IV did not account for the 10-fold difference of CV. By histochemical staining, it was revealed that VB to WM was heavily myelinated, whereas in the cortex staining became substantially weaker. We also found that such morphological and physiological characteristics developed in parallel and were accomplished around postnatal week 4. Considering that VB to WM is longer and more variable in length among afferents than is the intracortical region, such an enormous difference of CV makes conduction time heavily dependent on the length of intracortical region, which is relatively constant. Our finding may well provide a general strategy of connecting multiple sites irrespective of distances in the brain.

Long-term potentiation and N-methyl-D-aspartate receptors in the visual cortex of young rats.

1. Long‐term potentiation (LTP) of synaptic transmission following tetanic stimulation of the white matter was studied by recording extracellular field potentials and intracellular synaptic potentials from layer II/III of visual cortical slices from young rats ranging in age from 21 to 40 days. 2. Single shocks applied to the white matter at 0.1 Hz, used as test stimuli, elicited field potentials that consisted of primary and secondary components. The removal of Ca2+ ions from the perfusate allowed identification of the secondary component as originating postsynaptically and the primary one as reflecting a mixture of antidromic and postsynaptic potentials. 3. Tetanic stimulation at 5 Hz for 60 s was delivered to the white matter and field potentials were observed for 20 min to 9 h after the tetanus. LTP was defined as being present when the response displayed more than a 20% increase in amplitude of the Ca2+‐sensitive components 20 min after the tetanus. LTP was induced in twelve of twenty‐three slices tested, and this potentiation lasted throughout the period of observation. The average magnitude of potentiation was 147.8 +/‐ 28.4% of the control value for the twelve slices. 4. Administration of D,L‐2‐amino‐5‐phosphonovalerate (APV), an antagonist selective for N‐methyl‐D‐aspartate (NMDA)‐preferring receptors, slightly reduced the amplitudes of Ca2+‐sensitive components of the field potentials. The average magnitude of reduction was 80.2 +/‐ 15.3% of the pre‐drug control values. In the presence of APV, LTP was induced in only one slice of twelve tested. 5. Stable intracellular recordings were obtained from twenty‐three cells from layer II/III. Excitatory postsynaptic potentials (EPSPs) evoked by white matter stimulation had mean onset and peak latencies of 4.1 and 11.3 ms, respectively. In some cells these fast EPSPs were followed by another slow EPSP with a much longer latency and higher amplitude. Administration of APV revealed further that the fast EPSPs consisted of two components, i.e. early and late components. 6. Tetanization of the white matter induced long‐lasting enhancement of EPSPs in eight of twelve cells tested. In five of these eight cells, fast EPSPs were enhanced in amplitude and in the remaining three cells, slow EPSPs appeared de novo after the tetanus. 7. APV reduced the amplitudes of the fast EPSPs and abolished the slow EPSPs if present. The average magnitude of reduction for the fast EPSPs was 65.6 +/‐ 15.1% and this reduction was due mainly to an elimination of the late component.(ABSTRACT TRUNCATED AT 400 WORDS)

Excitatory amino acid transmitters and their receptors in neural circuits of the cerebral neocortex

In 1954, L-glutamate (Glu) and L-aspartate (Asp) were first suggested as being excitatory synaptic transmitters in the cerebral cortex. Since then, evidence has mounted steadily in favor of the view that Glu and Asp are major excitatory transmitters in the neocortex. Many of the experimental studies which reported how Glu/Asp came to satisfy the criteria for transmitters in the neocortex are reviewed here, according to the methods employed. Since the question of which particular synaptic sites in cortical neural circuits Glu/Asp operate as excitatory transmitters has not previously been reviewed, particular attention is given to efferent, afferent and intrinsic neural circuits of the visual and somatosensory cortices, where circuitry is relatively clearly delineated. Recent studies using chemical assays of released amino acids, high-affinity uptake mechanisms of Glu/Asp from nerve terminals, the direct micro-iontophoretic administration of Glu/Asp antagonists, and immunocytochemical techniques have demonstrated that almost all corticofugal efferent projections employ Glu/Asp as excitatory synaptic transmitters. Evidence indicating that thalamocortical afferent projections, including geniculocortical projections and some intrinsic connections are glutamatergic, is also reviewed. Thus, the results highlighted here indicate that the main framework of neocortical circuitry is operated by Glu/Asp. Pharmacological studies indicate that synaptic receptors for Glu/Asp can be classified into a few subtypes, including N-methyl-D-aspartate (NMDA) and quisqualate/kainate (non-NMDA) types. Some evidence indicating the sites of operation of NMDA and non-NMDA receptors in neocortical circuitry is reviewed, and the distinct, functional significance of these two types of Glu/Asp receptors in information processing in the neocortex is proposed.

Acetylcholine suppresses the spread of excitation in the visual cortex revealed by optical recording: possible differential effect depending on the source of input.

Optical recording with a voltage‐sensitive dye was performed in visual cortical slices of the rat to determine the effect of acetylcholine (ACh) on the spread of excitation. In the presence of ACh, the spread of excitation initiated by stimulation at the white matter/layer VI (WM/VI) was greatly suppressed throughout the cortex, with less suppression in the middle layers. By comparing the effect of ACh with that of 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), the fraction of the synaptic component that was sensitive to ACh was evaluated. ACh suppressed ∼ 40–50% (maximum 55.8%, n = 11) of the initial synaptic component in the superficial and deep layers. In the middle, however, the effect was weakest and only ∼ 20–30% (minimum 20.9%, n = 11) of the initial synaptic component was suppressed. On the basis of histological analysis, the region with the weakest ACh effect extended from upper V to lower II/III. To identify the site of ACh action in terms of pre‐ versus postsynaptic localization, exogenous glutamate was applied. Because ACh did not suppress the excitation induced by glutamate, the site of the ACh action was indicated to be presynaptic. When layer II/III was stimulated instead of WM/VI, the suppression was uniform throughout the cortex. A muscarinic receptor antagonist, atropine, blocked the suppression by ACh. In conclusion, our results indicate the following two points. First, ACh strongly suppresses intracortical connectivity through presynaptic muscarinic receptors. Secondly, in contrast to the intracortical connection, some group(s) of fibres, possibly thalamocortical afferents that arise from white matter and terminate in the middle cortical layers are suppressed much less by ACh. While ACh has been reported to have confusingly diverse effects, e.g. direct depolarization and hyperpolarization as well as synaptic facilitation and suppression, its effect on the propagation of excitation is very clear; suppression on intracortical connection, leaving thalamocortical inputs rather intact. We postulate that cholinergic innervation enables the afferent input to have a relatively dominant effect in the cortex.

Analysis of somatosensory evoked potentials to lateral popliteal nerve stimulation in man

Abstract Somatosensory evoked potentials (SEPs) to the lateral popliteal nerve were studied in 41 normal subjects. Analysis of the SEPs was carried out by observing amplitude distribution over the scalp, recovery function and alteration during sleep. 1. 1. The wave form of the SEPs was fairly consistent from subject to subject. The SEP had an initial positive peak (P1) and a negative peak (N1) at latencies of approximately 34 and 45 msec, respectively, and five alternative positive and negative peaks (P2, N2, P3, N3 and P4). For the sake of description, the response was divided into four components (components 1, 2, 3, and 4), each having the peak at P1, P2, P3 and P4, respectively. 2. 2. Scalp distribution of component 1 was restricted in the parietal area just posterior to the vertex, whereas that of component 2 was shifted more anteriorly. Component 3 had the widest distribution among all. Component 4 showed the next widest symmetrical distribution with the maximum at the vertex. 3. 3. After maximal excitation, components 1 and 2 recovered the control responsiveness at approximately 200 msec and 600 msec, respectively. Components 3 and 4 had much slower recovery processes. These components were still depressed even at 800 msec. 4. 4. When sleep progressed from stage 1 to stage 2 and further stage 3, component 1 was decreased without changing its peak latency, and the peak latency of component 2 was prolonged with an increase in amplitude. 5. 5. By comparison with the results of previous studies in animal and man, it was inferred the component 1 would be the postsynaptic potential of the primary response mediated by the specific thalamo-cortical projection system and would correspond to the component 2 of the medial nerve SEP as established by Allison (1962) and Goff et al. (1962). Component 2 may correspond to the association response of Amassian (1954). Component 3 may probably be related to the non-specific diffuse projection system. Component 4 may represent the positive phase of the V-potential.

Mechanisms underlying orientation selectivity of neurons in the primary visual cortex of the macaque.

1. Effects of blocking intracortical inhibition by microiontophoretic administration of bicuculline methiodide (BMI), a selective antagonist for GABAA receptors, on orientation selectivity of 109 neurones were studied in the primary visual cortex (V1) of anaesthetized and paralysed monkeys. 2. The averaged orientation tuning of visual responses of cells was poor in cytochrome oxidaserich blobs of layer II/III and in layer IVc beta, moderate in layers IVb, IVc alpha and V, and sharp in the interblob region of layer II/III and in layers IVa and VI. 3. Iontophoretic administration of BMI reduced the sharpness of orientation tuning of cells to a varying extent in each layer. In most cells, furthermore, the originally ineffective stimuli induced visual responses during the BMI administration, suggesting that excitatory inputs evoked by the non‐optimally oriented stimuli were masked by GABAergic inhibition. Nevertheless, the maximal facilitation was observed in the response to the optimally or near‐optimally oriented stimuli. 4. There was a difference in such an effect of BMI among layers. Orientation selectivity of cells in interblobs in layer II/III and in layer IVb was sensitive to BMI whereas that of cells in layer VI was relatively insensitive to BMI, suggesting a larger contribution of excitatory mechanisms to the orientation selectivity in this layer. 5. In the orientation‐selective cells, an analysis of the magnitude of excitation and inhibition evoked by stimuli at various orientations suggests that both inputs tune around the optimal orientation and their magnitudes are almost proportional to each other except at the optimal orientation. This analysis also indicates that the orientation tuning of inhibition had a less prominent peak around the optimal orientation than that of excitation. This dominance of excitation over inhibition around the optimal orientation may function to accentuate the response to the optimally oriented stimulus. 6. These results suggest that, in the monkey V1, the orientation selectivity of cells is largely dependent on the orientation‐biased excitatory and inhibitory inputs which have a broader tuning profile, covering from the optimal to null‐orientation, than that observed in extracellularly recorded responses at the control level.

Actions of excitatory amino acid antagonists on synaptic inputs to the rat medial vestibular nucleus: an electrophysiological study in vitro

SummaryThe actions of excitatory amino acid (EAA) antagonists on synaptic inputs to neurons in the rat medial vestibular nucleus (MVN) from ipsilateral vestibular afferents and vestibular commissures were studied in brain stem slice preparations. Antagonists used were 2-amino-5-phosphonovalerate (APV), a selective antagonist for the N-methyl-D-aspartate (NMDA) type of EAA receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a selective antagonist for the quisqualate/kainate (non-NMDA) type of EAA receptors and kynurenate (KYNA), a broad spectrum antagonist for the three types of EAA receptors. MVN neurons were classified as having mono- or polysynaptic inputs from vestibular afferents and commissural fibers by calculating synaptic delay. An application of APV through the perfusion medium suppressed 82% of cells activated monosynaptically from commissures, while it suppressed only 9% of cells activated monosynaptically from vestibular afferents. The application of KYNA proved much less selective, suppressing 83% of the former group of cells and 93% of the latter. CNQX suppressed almost all the cells of both groups. The sensitivity of monosynaptic inputs to KYNA, CNQX or APV was not significantly different from that of polysynaptic inputs irrespective of sources of inputs. These results suggest that excitatory synaptic inputs to MVN neurons are mediated mainly through non-NMDA type of EAA receptors from vestibular afferents and through NMDA as well as non-NMDA types of EAA receptors from commissures.

Pyramidal tract control over cutaneous and kinesthetic sensory transmission in the cat thalamus

SummaryIn the thalamic ventrobasal complex (VB) of the cat, effects of electrical stimulation of the pyramidal tract (PT) upon activities of 112 relay cells and 18 internuncial cells were examined. Single PT shocks to the cerebral peduncle elicited short-latency discharges in 31 relay cells (mean latency, 1.4±0.5 msec). When weak PT stimuli were employed as conditioning shocks, facilitatory effects upon responses to medial lemniscal (ML) stimulation were observed. It was revealed that VB relay cells were excited monosynaptically via collaterals of the fast PT fibers. Among 31 PT-excited cells 22 were fired by movements of joints (joint-movement units) and they made up 88% of all the joint-movement units. A majority of the relay cells responding to stimulation of hairs (hair units) did not receive excitatory effects from PT, except some special ones which represented long hairs at the distal or proximal end of the forearm-forepaw.In 44 relay cells repetitive PT shocks suppressed both evoked responses to ML stimulation and spontaneous discharges for 70–100 msec. Of these, 34 were hair units. The PT-induced inhibition in the hair units increased as their receptive fields shifted from the trunk towards the digits. Some intracellular recordings showed that the PT-induced inhibition was due to IPSPs generated disynaptically.Among 18 interneurons presumed to be inhibitory 10 responded with short latencies to PT stimulation. These were mostly the interneurons which presumably subserve the recurrent collateral inhibition in VB.

A Local Reduction in Cortical GABAergic Synapses after a Loss of Endogenous Brain-Derived Neurotrophic Factor, as Revealed by Single-Cell Gene Knock-Out Method

To address questions of whether brain-derived neurotrophic factor (BDNF) released from active excitatory neurons acts locally only on GABAergic presynaptic terminals contacting these neurons or generally also on GABAergic terminals contacting other inactive neurons, we developed a single-cell gene knock-out method in organotypic slice culture of visual cortex of floxed BDNF transgenic mice. A biolistic transfection of Cre recombinase with green fluorescence protein (GFP) plasmids to layer II/III of the cortex resulted in loss of BDNF in a single neuron or a small number of neurons, which expressed GFP at 13–14 d in vitro. Analysis with in situ hybridization and immunohistochemistry confirmed that neurons expressing GFP lacked BDNF mRNA and protein, respectively. Analysis with immunohistochemistry using antibody against GABA synthesizing enzyme showed that the number of GABAergic terminals on the soma of BDNF knock-out neurons was smaller than that of neighboring control neurons. Morphological analysis indicated that there was no significant difference in the soma size and branch points and length of dendrites between the BDNF knock-out and control neurons. Recordings of miniature IPSCs (mIPSCs) showed that the frequency of mIPSCs of BDNF knock-out neurons was lower than that of control neurons, although the amplitude was not significantly different, suggesting the smaller number of functional GABAergic synapses on whole the BDNF knock-out neuron. The present results suggest that BDNF released from postsynaptic target neurons promotes the formation or proliferation of GABAergic synapses through its local actions in layer II/III of visual cortex.