Department of Physiology, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK
Address correspondence to Alex M. Thomson, Department of Physiology, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK. Email: alext{at}ucl.ac.uk.
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Abstract |
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Introduction |
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To provide more detail about these local circuit connections, a number of studies have employed paired intracellular recordings to document the probabilities with which pairs of neocortical neurons make synaptic connections within the same layer and between layers and the properties of those synaptic connections. For example, connections between pairs of layer 5 pyramidal cells in the immature (Lübke et al., 1996; Markram and Tsodyks, 1996
; Markram et al., 1997
) and mature rat neocortex (Thomson et al., 1993a
; Thomson and Deuchars, 1997
) are less frequent and typically involve pairs of neurons that are closer neighbours, but often generate EPSPs (excitatory postsynaptic potentials) of larger amplitude than are typical for pairs of pyramidal cells in layer 3 (Thomson and Deuchars, 1997
; Reyes and Sakmann, 1999
).
A very high probability input to large, tufted layer 5 pyramidal cells comes from layer 3 pyramidal cells whose somata are within 100 µm of the postsynaptic apical dendrite in adult (Thomson and Bannister, 1998) and immature rat neocortex (Reyes and Sakmann, 1999
). In contrast, little (1:58, adult) or no (immature) input in the opposite direction, from layer 5 pyramids to layer 3 pyramids, was found. There was, however, a significant excitatory input from layer 5 to layer 2/3 interneurons with an adapting firing pattern, while fast spiking interneurons and pyramids in layers 2/3 received their strongest inputs from cells in the middle layers (Dantzker and Callaway, 2000
). These data demonstrate the layer-specific selectivity with which pyramids choose their targets. The forward flow of information (e.g. layer 3 to 5) involves a strong excitatory input to just one class of pyramids as well as to interneurons whose axonal arbours can ramify in both layers 5 and 3 (Thomson et al., 1996
). In contrast, the return pathway (layer 5 to 3) appears to select layer 3 interneurons of a particular class, avoiding other interneuronal and pyramidal targets in that layer. An obvious question that arises is whether the forward pathway from layer 4 to layer 3 and any return connection from layer 3 to layer 4 neurons exhibit similar preferences.
Within single barrels in the barrel fields of the immature rat somatosensory cortex (Feldmeyer et al., 1999a; Gibson et al., 1999
; Petersen et al., 2000) and adult cat visual cortex (Stratford et al., 1996
) synaptic connections between layer 4 excitatory, spiny neurons (both spiny stellate and pyramidal cells) have been reported to occur in 1130% of tested pairs of neurons. Probabilities of connections between inhibitory and excitatory neuron pairs in this layer were around 9% in adult cat visual cortex (Tarczy-Hornoch et al., 1998
), while in the immature rat 41% of regular spiking layer 4 neurons tested elicited EPSPs in simultaneously recorded interneurons and a very high probability of connectivity (both chemical and electrical) between pairs of interneurons was observed in immature layer 4, particularly where the two cells displayed similar electrophysiological characteristics (Galarreta and Hestrin, 1999
; Gibson et al., 1999
).
The strong input from layer 4 to layer 3 that might be predicted from anatomical studies has not been as thoroughly documented with paired recordings to date. In the immature rat neocortex the connectivity underlying the excitatory input from layer 4 to layer 3 pyramidal cells was reported to be remarkably high, although no ratios are given and the inputs were relatively weak, involving small EPSPs (Feldmeyer et al., 1999b, 2000). The possibility exists, however, that this pathway develops its full potential only after thalamo-cortical inputs and layer 4 connectivity have matured. No descending excitatory connections from layer 3 to layer 4 were found and connections between these layers involving inhibitory interneurons were not observed. Since layer 3 pyramidal axons rarely ramify in layer 4, the lack of such a back projection is precisely what might be expected for targets whose dendrites are confined to layer 4, i.e. small interneurons and spiny stellate cells. However, layer 4 also contains pyramidal cells and larger interneurons whose dendrites project into layer 3. Do layer 3 pyramidal axons therefore recognise layer 4 pyramids as inappropriate targets and distinct from layer 3 pyramids? One excitatory connection from a layer 3 pyramidal cell to a dendrite-targeting interneuron in layer 4 has been described in the adult cat (Buhl et al., 1997
), but the relative density of such connections was not determined.
The majority of paired recordings in neocortical slices have used rat and many more recent studies have used immature preparations in which the fine details of cortical circuitry may still be developing. However, a large body of in vivo work describing the response properties of and predicting functions for neocortical neurons and networks uses adult cats and primates. One question addressed in this study was, therefore, the degree to which some of the simplest building blocks of the local circuit in adult rat and cat can be considered comparable. For example, where a synaptic connection of a given type is observed to occur in either a high or a very low proportion of recorded pairs in one species, does it occur and with equivalent probability in the other and are the synaptic events elicited comparable? Paired and triple intracellular recordings were therefore made in slices of neocortex obtained from adult rats and cats. Synaptically connected neurons were filled with biocytin and cells identified after histological processing. Synaptic connections between layers 3 and 4 were of particular interest, but to allow comparison between species and with previous studies, the characteristics of other connections that have been more thoroughly documented in the rat were included.
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Materials and Methods |
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Young adult male SpragueDawley rats (120160 g) were anaesthetized with inhaled Fluothane (AstraZeneka, Luton, UK) then 60 mg/kg i.p. sodium pentobarbitone (Sagatal; Rhone Merieux, Harlow, UK) and perfused transcardially with 50100 ml ice-cold artificial cerebrospinal fluid (ACSF) with added sodium pentobarbitone (60 mg/l). This modified ACSF contained 248 mM sucrose, 25.5 mM NaHCO3, 3.3 mM KCl, 1.2 mM KH2PO4, 1.0 mM MgSO4, 2.5 mM CaCl2 and 15 mM D-glucose equilibrated with 95% O2/5% CO2. The animals were decapitated, the brain removed and 450500 µm coronal sections of brain including the neocortex cut (Vibroslice; Campden Instruments, UK). Slices were maintained at the interface between the sucrose-containing ACSF (without pentobarbitone) and warm, humidified 95% O2/5% CO2 at 3536°C for 1 h. The sucrose-containing medium was then replaced with a standard ACSF in which 124 mM NaCl replaced the sucrose. This ACSF was used for all recordings, which commenced after another hour.
Slices of cat neocortex were obtained from animals anaesthetized for a different series of acute experiments reported elsewhere (Wang et al., 2000, 2002
). Young adult male cats (2.53.4 kg) were anaesthetized with a mixture of
-chloralose (70 mg/kg) and pentobarbitone sodium (6 mg/kg) injected i.v. The right carotid artery was cannulated and the skull overlying the occipital lobes removed. The animal was killed with an overdose of i.v. barbiturate (100 mg/kg), the left carotid artery tied off and the two jugular veins cut prior to perfusion through the right carotid with 200 ml of ice-cold modified ACSF (sucrose-containing). The dura overlying the visual cortex was cut and peeled back and a block including visual cortex removed. Slice cutting, maintenance and recording procedures were then identical to those used for the rat brain slices. All procedures complied with British Home Office regulations for animal use.
Electrophysiological Recordings
Paired and triple intracellular recordings were performed using conventional sharp electrodes (80160 M) containing 2 M potassium methylsulphate and 2% biocytin (w/v) under current clamp (AxoProbe and AxoClamp; Axon Instruments, CA). Sharp electrodes are more efficient for multiple recordings in thick slices of adult tissue which maintain the integrity of large neurons and local circuitry. Typically, a single stable intracellular recording was obtained in a previously unstudied region of the slice. A second electrode was then introduced and up to six other cells sampled until a synaptic connection was observed. Presynaptic firing was initiated by intracellular current injection at 1 pulse/3 s. Pulses were combinations of square wave and ramped currents. A third electrode was then introduced into a neighbouring area or layer, but one that had not previously been sampled. Again, neurons were sampled with this third electrode until a connection with one or both of the other neurons was found or until recordings deteriorated. In some experiments, after a triplet of connected cells had been recorded, one of the electrodes was withdrawn and a search made for other connected neurons. A map was drawn of the recorded region and the position of each sampled cell marked to aid subsequent identification of recorded neurons after histological processing. Continuous analogue recordings from presynaptic and postsynaptic neurons were made on analogue tape (Racal, Southampton, UK). Data from the longer and more detailed protocols in this series of experiments involving the frequency-dependent properties of the synaptic connections will be reported elsewhere.
Data Analysis
Data were digitized (510 kHz, voltage resolution 0.0050.01 mV) and analysed off-line (Spike 2 data collection and in-house analysis software). Individual sweeps were observed and trigger points associated with the rising phase of each presynaptic action potential (AP) checked/edited. Sweeps including large spontaneous events or artifacts were rejected. Averaging of EPSPs and IPSPs was triggered by the rising phase of single presynaptic spikes for the average of the first EPSP or IPSP, the rising phase of the second presynaptic spike for the average second EPSP/IPSP in a train and so on. Some of the illustrated EPSP/IPSPs are therefore composite averages with single spike responses and second (third, ...) EPSP/IPSPs elicited at specific interspike intervals superimposed (individual averages in these composites include 20200 sweeps). Average first or single spike EPSP/IPSP amplitudes were measured as the difference between an average of the voltage preceding the presynaptic spike and an average around the peak of the EPSP/IPSP. The 1090% rise time (RT) and width at half amplitude (HW) were measured from averaged single spike EPSP/IPSPs.
Histological Processing
Recorded cells were filled with biocytin, either by passive diffusion from the recording electrodes or by passing positive current in a half duty cycle (0.5 nA, 500 ms, 1 pulse/s). Slices were fixed overnight in 0.1 M phosphate buffer containing 2.5% glutaraldehyde, 4% paraformaldehyde and 0.5% saturated picric acid, then washed in 0.1 M phosphate buffer. They were then embedded in gelatin, sectioned at 60 µm (Vibratome), cryoprotected in 30% sucrose plus 12% glycerol and permeabilized by freezethawing above liquid nitrogen. Biocytin was localized using the Elite ABC kit (Vector) overnight and visualized using 3',3'-tetraminodiaminobenzidine (DAB) (Sigma) intensified with nickel chloride. Sections were then dehydrated and embedded in Durcupan resin (Fluka) on slides. Filled neurons were reconstructed using a drawing tube (100x objective, 1000x magnification).
Some of the slices in which interneurons of interest had been recorded were fixed in a low gluteraldehyde solution (4% paraformaldehyde, 0.5% picric acid plus 0.025% gluteraldehyde) and first processed for immunofluorescent identification of cellular markers in biocytin-filled cells, before permanent peroxidase labelling of filled neurons, as above. Details of immunofluorescence protocols and the cocktails of antibodies used can be found in a previous publication (Hughes et al., 2000). Typically, avidin labelled with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) was used to visualize the biocytin. A fluorescein isothiocyanate (FITC)-labelled secondary antibody was used to identify a monoclonal mouse antibody and a Texas red-labelled secondary used to visualize a polyclonal rabbit antibody. Most commonly in this series, a monoclonal antibody directed against parvalbumin (clone PA-235; Sigma, Poole, UK) was used with a polyclonal antibody raised against another marker, such as calbindin (R8701 or R9501, K. Baimbridge). In some cases a mouse monoclonal antibody raised against gastrin/CCK (cholecystokinin) was used (no. 9303 CURE; UCLA). For these protocols to be successful, i.e. for the filled cells to be identifiable with AMCA fluorescence, strong labelling of recorded neurons with biocytin was required. For unambiguous identification of cell markers, full penetration of the tissue by the primary and the labelled secondary antibodies was also necessary, and in some sections this was not achieved. The monoclonal antibodies typically resulted in more complete penetration without additional permeabilization of the tissue. Cells are therefore reported as immuno-positive or immuno-negative for a given marker where the interneuron was identified with AMCA fluorescence and where FITC and/or Texas red fluorescence in that cell, and/or in adjacent cells at the same focal depth, demonstrated adequate penetration of the antibodies.
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Results |
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Of 81 pairs of layer 2/3 pyramidal cells tested in cat visual cortex (54 in area 17, 20 in area 19 and seven in area 18), seven were connected, two in area 19 and five in area 17, of which one was reciprocal (eight EPSPs, connectivity ratio 1:10, Figs 1 and 4). This includes two pairs in which the presynaptic pyramid was in layer 3 and the postsynaptic in layer 2 (Fig. 2
). With the exception of one unusually large EPSP, the average amplitudes of the EPSPs recorded in cat layer 2/3 were similar to those recorded in the rat (Table 1
). Mean EPSP duration was longer in cat, but as the range was broad, the difference was not significant (P > 0.05). The largest EPSP (8.2 mV) in this group is listed separately in Table 1
and involved two large layer 3 pyramids at the border with layer 4. In cat, as described previously in rat, pyramidpyramid EPSPs typically exhibited paired pulse and frequency-dependent depression throughout trains of presynaptic spikes at high frequency (
100 Hz) (Thomson, 1997
) (Fig. 1
). In some, modest facilitation of the second EPSP could occur at slightly longer interspike intervals, after recovery from the shortest interval depression (Fig. 2
). However, even in these connections, third and subsequent EPSPs in trains typically exhibited depression throughout the range of interspike intervals studied (580 ms). More detailed analysis of responses to spike trains of different frequencies is reported elsewhere.
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PyramidPyramid Connections in Layers 2/3 and 5 of Rat Neocortex
The data obtained in rat (Table 1) confirm previous reports that the connectivity between neighbouring pyramidal cells in layers 2/3 is higher than in layer 5 and that the descending projection from layer 3 pyramids to large, tufted layer 5 pyramids is strong, largely unidirectional and tightly focused. The electrophysiological properties of layer 3 pyramidpyramid EPSPs are similar in rat and cat. In the cat, however, a lower connectivity ratio was observed than in the rat (1:10 compared with 1:4). It should perhaps be noted, however, that the ratios observed here in adult rat layer 2/3 are considerably higher than our earliest studies in this species [e.g. 1:38 (Thomson and West, 1993
)]. This probably reflects the development of more efficient search strategies over time and the use of thicker slices (450500 compared with 400 µm) in our more recent studies. In the present study, synaptically connected layer 2/3 pyramidal cell pairs in rat were typically more widely separated in the horizontal plane (47 ± 37 µm) than were layer 5 pyramidal pairs (17 ± 12 µm), but in the cat, synaptically connected layer 2/3 pyramidal pairs were on average more widely separated again (69 ± 44 µm). The use of a similar search strategy in the two species may have introduced some bias in favour of finding connected pairs in the rat neocortex where neurons are, on average, smaller.
Synaptic Connections Between Layer 4 Excitatory Cells in Cat Visual Cortex
Of 23 pairs of excitatory neurons in cat layer 4, four pairs yielded an EPSP, an average connectivity ratio of 1:5.7, which is similar to previous observations in immature rat (Feldmeyer et al., 1999a,b
; Gibson et al., 1999
) and adult cat (Stratford et al., 1996
). Two of these connections were confirmed histologically and involved spiny stellate to spiny stellate connections. In one of these examples both layer 4 spiny stellate cells also activated EPSPs in a single layer 3 pyramidal cell (see Fig. 3A
). Too few paired recordings were made in layer 4 of the rat neocortex to produce estimates of connectivity ratios. In the one connected pair confirmed histologically in rat, both layer 4 cells were pyramidal cells (Fig. 3B
).
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In cat, seven of 70 such pairs yielded an EPSP (1:10, all in area 17). Of the five pairs confirmed histologically, four involved pre-synaptic spiny stellate cells in middle to upper layer 4 (Fig. 3B). The EPSPs elicited by middle to upper layer 4 cells were similar in amplitude, but briefer on average than those elicited by other layer 3 pyramidal cells. Smaller, slower EPSPs were, however, elicited in layer 3 pyramidal cells by two deep layer 4 cells (one identified as a deep layer 4 pyramid). These EPSPs had average amplitudes of 0.2 and 0.15 mV, 1090% rise times of 5 and 4 ms and widths at half amplitude of 53 and 50 ms at membrane potentials of 78 and 70 mV, respectively (Fig. 4
). For a closer comparison with data obtained in the rat in which all presynaptic layer 4 cells were in middle to upper layer 4 the measurements of these two EPSPs are not included in the means in Table 1
.
Excitatory Connections From Layer 4 to Pyramids in Layer 3 of Rat Neocortex
Of 25 such pairs tested in somatosensory and visual cortex, seven yielded an EPSP (1:3.6). All connections were ascending, i.e. from layer 4 to layer 3. Three pairs were confirmed histologically. In rat the majority of connected pairs appeared to involve layer 4 pyramidal cells as in this series no spiny stellate cells, but a large number of layer 4 pyramidal cells, were recovered histologically in rat neocortex. Only two of these EPSPs were stored and measured (Table 1). The others were brief recordings, but they were typically noted to be
2 mV in average amplitude and in rat larger than EPSPs elicited by other, simultaneously recorded presynaptic layer 3 pyramids.
In cat the EPSPs elicited by cells in middle to upper layer 4 were on average similar in duration but smaller than those observed in rat and the connectivity ratios were higher in rat (Table 1). However, the probabilities of a layer 3 pyramidal cell receiving an input from another layer 3 pyramidal cell and from a layer 4 excitatory cell were approximately equal in each of the two species (1:4 compared with 1:3.6 in rat and 1:10 compared with 1:10 in cat). All connections were ascending, i.e. no EPSP was elicited in a layer 4 excitatory neuron by APs in a layer 3 pyramidal cell in these 94 pairs. Connections between layer 4 excitatory cells (ratio 1:5.7, cat) can in principle be in either direction, while the excitatory connection between layer 4 and layer 3 is essentially unidirectional. The probability of any single layer 4 excitatory cell innervating another layer 4 excitatory cell and innervating a deep layer 3 pyramidal cell would therefore appear to be similar.
Morphology of Labelled Interneurons
Forty seven interneurons were stained sufficiently well in cat visual cortex for identification, two in layer 2, 16 in layer 3, 27 in layer 4 and two in layer 5. Small to medium sized multipolar interneurons whose dendrites and often dense axonal arbours were confined to the layer of origin were found in layers 25 (one in layer 2, six in layer 3, six in layer 4 and one in layer 5). Some of these cells resembled cells described previously as small to medium sized basket cells, but no ultrastructural analysis of the synapses they formed was done to confirm this and their dendritic and axonal arbours indicated a non-homogeneous population. Five of these cells were immuno-positive for parvalbumin.
Six neurogliaform cells were recovered (one in layer 2, one in layer 3 and four in layer 4), but no connections involving these cells could be unambiguously confirmed. Other interneurons whose processes were largely confined to the layer of origin included six cells with flask-shaped somata (one in layer 3 and five in layer 4) from which the axon and all the major dendrites exited one pole in a tuft with only a few very short dendrites issuing from other parts of the soma. In deep layer 4 (three cells) the main dendrites of these cells exited the more superficial pole and extended through the depth of the layer. In middle and upper layer 4 (two cells) and in upper layer 3 the main dendrites exited the deeper pole and extended to the lower border of the layer. The axons of two of these flask-shaped cells formed long almost straight collaterals, radiating vertically and diagonally out from the soma and terminating in discrete arbours 100200 µm away (one such arbour originating in layer 4 innervated lower layer 3), while the axon of another such cell formed a dense, vertically oriented arbour that spanned layer 4, with a single major branch ascending into lower layer 3, looping and descending again to ramify in layer 4 (Fig. 11). Finally, a small multipolar neuron in middle to upper layer 4 that targeted more distal dendrites of its single identified postsynaptic neuron was recovered.
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The interneurons filled in the rat cortex in this study did not display such a wide range of sizes or morphological characteristics, although a range of morphologies similar to that observed here in cat has been reported in rat (Kawaguchi and Kubota, 1997; Gupta et al., 2000
), including several subclasses of basket cells (Wang et al., 2002
) and VIP immunoreactive bipolar cells with vertically oriented axonal arbours (Rozov et al., 2001
). Most of the recovered cells in the present study were multipolar and had axons and dendrites confined to the layer of origin (or to layers 2 and 3). Four of the layer 3 interneurons recovered are illustrated in Figure 5
, with the connections recorded.
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Within Layer Connections Between Pyramidal Cells and Interneurons in Layer 2/3 of the Cat Visual Cortex
In layers 2/3 of the cat visual cortex, 25 such pairs were recorded (12 interneurons). Six of these pairs yielded EPSPs (five interneurons, 1:4) and seven yielded IPSPs (1:3.6) (three of which were reciprocal connections). All but one of the EPSPs recorded in cat interneurons were depressing, i.e. the EPSPs declined in average amplitude with successive APs in trains (Figs 6 and 11). More detailed analysis of these events will be published elsewhere.
The layer of origin and interneuronal soma/dendritic morphology were confirmed for all 12 interneurons, but some yielded only partial axonal recoveries. The postsynaptic interneuron for the one facilitating EPSP recorded in cat cortex was, for example, only partially recovered. The dendritic and axonal arbours of three of the well-labelled presynaptic layer 3 interneurons were restricted to the superficial layers (two were reciprocally connected with layer 3 pyramidal cells). Two of these, a multipolar and a flask-shaped internerneuron innervated both layer 3 and layer 2. The third, which resembled a small basket cell, appeared only to innervate layer 3. A fourth interneuron that was reciprocally connected with a layer 3 pyramidal cell had a sparsely spiny, bi-tufted dendritic tree extending into layer 4. All its axon ascended, with many fine, vertical collaterals coursing to layer 1, forming small clusters of boutons en route in all three layers and forming close membrane appositions with the distal dendrites of the pyramidal cell (a Martinotti-like cell, Fig. 7). The reciprocal connection involving this cell was, however, only briefly recorded and the recording is not illustrated.
Several layer 3 interneurons innervated both layer 3 and layer 5. The axon of one large (parvalbumin immuno-negative) layer 3 interneuron, in addition to generating a dense local arbour and major myelinated axonal branches that extended horizontally for more than 1 mm, had a single myelinated branch descending through layer 4 to layer 5, where it generated a second, much narrower arbour (Fig. 6). This interneuron was reciprocally connected with a smaller, layer 2 interneuron and received EPSPs from a layer 3 pyramid and from three layer 4 excitatory cells (partial recoveries). The dendrites of another presynaptic layer 3 interneuron were too weakly stained for reconstruction, but the axon was well labelled. It also generated a dense local arbour as well as long horizontal branches and sent four vertical axon collaterals, in a narrow bundle, down through layer 4 to layer 5. This axon made six close membrane appositions with primary and secondary dendrites of a layer 3 pyramid in which it elicited an IPSP 1 mV in amplitude (at 63 mV). It thus resembled the double bouquet cells described previously (see for example Tamás et al., 1997
). Another small incompletely recovered layer 3 interneuron, with a more accommodating firing pattern than a classical fast spiking cell, was reciprocally connected with two layer 3 pyramidal cells and generated an IPSP in a layer 5 cell. Thus, while some layer 3 interneuronal axons are restricted to layer 3, others inhibit both layers 3 and 5 (see also Buhl et al., 1997
). In this series, no layer 3 interneuron in the rat that demonstrably innervated layers 3 and 5 was recovered, but double bouquet cells have also been described in this species (Kawaguchi and Kubota, 1997
).
Within Layer Connections Between Excitatory and Inhibitory Neurons in Cat Layer 4
In 42 paired recordings in layer 4, one cell was an excitatory neuron (a pyramidal or spiny stellate cell) and the other an inhibitory interneuron (17 interneurons). Eight of these pairs yielded excitatory connections (five interneurons, including one reciprocal connection), giving a ratio of 1:5, similar to that observed in layer 3. The five postsynaptic interneurons were all multipolar cells, three with dendrites confined to layer 4. In one of these examples a spiny stellate cell innervated a parvalbumin immuno-positive interneuron (whose axon could not be reconstructed) as well as another spiny stellate cell (Fig. 8). Two of the postsynaptic interneurons had dendrites extending into layer 3 and also received excitatory input from layer 3 pyramidal cells. One of these was a large cell (CCK immuno-negative) which generated a very wide axonal arbour spanning the border between the two layers (Fig. 11
). The axon of the other cell, a smaller multipolar interneuron, descended through the depth of layer 4 and into layer 5.
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Within Layer Connections Between Pyramidal Cells and Interneurons in Layers 2/3 of the Rat Neocortex
In layers 2/3 of the rat neocortex, 107 pairs in which one cell was a pyramidal cell and the other an interneuron (25 interneurons) were recorded. Of these, 22 pairs yielded an excitatory (1:5) and 17 an inhibitory connection (1:6.3) (including three reciprocal connections). Four of the EPSPs were strongly facilitating, i.e. the first AP of a train resulted in a small EPSP (or no discernable postsynaptic response), but successive APs elicited EPSPs of steadily increasing amplitude. Three of these facilitating EPSPs were recorded in a single interneuron and one in another (these EPSPs are excluded from the means in Table 1). The remaining layer 3 pyramid to interneuron connections resulted in depressing EPSPs (Fig. 5A,C,D
). With any one postsynaptic interneuron the frequency-dependent effects were consistent, i.e. if the input from one pyramidal cell was strongly facilitating, the inputs from any other presynaptic excitatory cells studied were also strongly facilitating, and likewise for depressing EPSPs (see for example Fig. 5A
). Four of the interneurons receiving depressing EPSPs were immuno-positive for parvalbumin (Fig. 5C,D
), two were immuno-negative for parvalbumin and three were not successfully tested by immunofluorescence (Fig. 5A
).
Comparing the two species, the probability, within these samples, of a layer 3 pyramidal cell innervating a neighbouring layer 3 interneuron was similar in rat (1:5) and cat (1:4), while the probability of a layer 3 interneuron inhibiting a neighbouring layer 3 pyramidal cell was higher in cat than in rat (1:3.6 compared with 1:6.3).
Within Layer Connections Between Pyramidal Cells and Interneurons in Layer 5 of the Rat Neocortex
In layer 5 of the rat neocortex, 73 pairs in which one neuron was a pyramidal cell and the other an inhibitory interneuron were recorded. Of these, seven exhibited an excitatory connection (1:10.4) and nine an inhibitory connection (1:8). These connectivity ratios are similar to those described for equivalent connections in previous studies. The EPSPs were largely depressing, although one showed modest facilitation (30%) of the second EPSP but depression of succeeding EPSPs in the train. The small circuits studied with triple recordings that were confirmed histologically involved one sparsely spiny interneuron that received an EPSP from a layer 5 pyramid and elicited IPSPs in three other L5 pyramids and one smooth parvalbumin immuno-positive interneuron that received one EPSP from a L5 pyramid, an IPSP from another (unidentified) L5 interneuron, with which it was reciprocally connected and elicited IPSPs in two other L5 pyramids.
Excitatory Inputs From Layer 4 to Layer 3 Interneurons
In 31 pairs recorded in cat, one neuron was an excitatory cell in layer 4 and the other an inhibitory interneuron in layer 3 (nine interneurons). In these 31 pairs three EPSPs were recorded, which would give a ratio of 1:10. All three EPSPs were, however, recorded from a single interneuron. This was the largest layer 3 interneuron recovered, the large parvalbumin immuno-negative interneuron (described above) whose dendrites extended into layer 4 and which generated a wide axonal arbour in layer 3 and a narrow arbour in layer 5 (Fig. 6). The three EPSPs elicited in this interneuron by the three presynaptic layer 4 cells were similar (amplitude 0.91.5 mV, RT 0.70.8 ms, HW 57.5 ms) and smaller than the one EPSP elicited in this interneuron by a layer 3 pyramidal cell (3.9 mV, RT 0.8 ms, HW 4.7 ms). These data suggest that only a minority of layer 3 interneurons, perhaps only the largest, are activated by layer 4 cells, but that those that are activated receive a strong ascending drive. This is, however, too small a sample from which to extrapolate far. In rat, 10 similar pairs were studied (five interneurons), only one of which yielded a small EPSP (<0.2 mV average amplitude). None of the 41 pairs tested resulted in an inhibitory connection from a layer 3 interneuron to a layer 4 excitatory cell.
Inhibitory Connections From Layer 4 Interneurons to Layer 3 Pyramidal Cells
Of 37 pairs in cat visual cortex in which a layer 4 interneuron was recorded simultaneously with a layer 3 pyramidal cell (15 interneurons), 10 yielded inhibitory connections. This gives a connectivity ratio (1:3.7) similar to inhibitory to excitatory cell connections within layer 3 in cat (1:3.6). This is higher than inhibitory to spiny cell connections within layer 4 (1:10) in this and a previous study in adult cat (Stratford et al., 1996; Tarczy-Hornoch et al., 1998
), but lower than in studies of intra-laminar connections in immature rat layer 4 (Galarreta and Hestrin, 1999
; Gibson et al., 1999
).
Six of these layer 4 interneuron to layer 3 pyramid pairs (involving five interneurons) were confirmed histologically. These interneurons had axonal arbours that innervated both layer 3 and layer 4. A bipolar interneuron in upper layer 4 inhibited a spiny stellate cell and another interneuron in layer 4 as well as a deep layer 3 pyramidal cell, making close membrane appositions with second order spiny postsynaptic dendrites (Fig. 9). A large CCK immuno-positive interneuron in layer 4, with a dense local axonal arbour in upper layer 4 and layer 3, long horizontal projections and a narrow descending projection to layer 5, innervated a layer 3 pyramid, making close membrane appositions with its fourth and fifth order dendrites (Fig. 10
). Two of the layer 4 interneuron to layer 3 pyramid IPSPs originated from a single large CCK immuno-negative basket cell, which innervated lower layer 3 and layer 4 and had long horizontal axonal projections. It made contact with the somata and very proximal dendrites of its postsynaptic layer 3 targets. This cell also inhibited a small flask-shaped deep layer 4 interneuron and received excitatory inputs from another layer 3 pyramid and from several unstable layer 4 excitatory cells, as well as an inhibitory input from another small flask-shaped interneuron in deep layer 4 (Fig. 11
). Thus interneurons whose somata are in layer 4 but which also inhibit layer 3 pyramidal cells include both basket cells that target very proximal portions of pyramidal cells and interneurons that innervate pyramidal dendrites.
Of 12 pairs in rat neocortex in which a layer 4 interneuron was recorded simultaneously with a layer 3 pyramidal cell (five interneurons), six yielded inhibitory connections. This also yields a high connectivity ratio (1:2), suggesting that while layer 3 interneurons rarely inhibit layer 4 spiny cells in either species, with the exception perhaps of layer 3 double bouquet cells, layer 4 interneurons of a variety of subclasses provide significant inhibition to lower layer 3 pyramidal cells in both species.
Excitatory Inputs from Layer 3 Pyramidal Cells to Layer 4 Interneurons in Cat Visual Cortex
Of the 37 such pairs recorded (15 interneurons), seven yielded an EPSP (1:5.3). The postsynaptic interneurons were in the upper half of layer 4 and most had both dendrites and axons that ascended into layer 3. Four of these cells that were successfully tested with immunofluorescence for parvalbumin were found to be parvalbumin immuno-positive and (like the CCK immunonegative cell) were either classical fast spiking or late spiking, fast spiking cells, with an interrupted or stuttering firing pattern. One of these interneurons was a basket cell with an intermediate sized axonal arbour that projected upwards and ramified predominantly in layer 3 where it was reciprocally connected with a pyramidal cell, while another (whose axon could only be partially reconstructed) sent a horizontally oriented axon away from its soma in layer 4 and had a major vertical branch that descended to layer 5. The CCK immunonegative layer 4 basket cell that was postsynaptic to a layer 3 pyramidal cell (and presynaptic to two others) had a broad axonal arbour, innervating upper layer 4 and lower layer 3 (Fig. 11).
The resultant EPSPs recorded in the parvalbumin immunopositive cells were the briefest recorded (RT 0.70.8 ms, HW 4.46.4 ms, amplitudes 0.71.7 mV). The EPSP in the CCK immuno-negative cell was of slightly longer duration (RT 0.7 ms, HW 9 ms, amplitude 1.23 mV). All these EPSPs were depressing (Fig. 11). Since the connectivity ratio (1:5.3) was similar to that for pyramid to interneuron connections within layer 3 in the cat (1:4), layer 3 pyramidal axons do not appear to distinguish between interneuronal targets whose somata are in layer 3 and those whose somata are in mid to upper layer 4, although they appear to avoid making contact with the apical dendrites of layer 4 pyramidal cells. In the rat 12 such pairs were recorded, one of which yielded an EPSP. Clearly this is too small a sample to allow a comparison, but demonstrates that a similar connection exists in both species.
Interneuron to Interneuron Connections
In eight paired recordings in rat layer 3 both cells were interneurons. These pairs yielded two IPSPs (ratio 1:4). In one example, a parvalbumin immuno-positive interneuron received, from an unidentified interneuron, an IPSP that was 2 mV average amplitude at 68 mV (RT 2 ms, HW 8 ms). This postsynaptic interneuron was also reciprocally connected with a layer 3 pyramidal cell in which it generated a slower IPSP. Of five interneuroninterneuron pairs tested in rat layer 5, two were connected (one reciprocal, three IPSPs, ratio 1:1.7).
In the only interneuroninterneuron pair recorded in layer 2/3 in the cat, a small layer 2 interneuron was reciprocally connected with a large (parvalbumin immuno-negative) layer 3 interneuron (amplitudes 0.8 and 1.3 mV, RT 2.3 and 3.9 ms, HW 7 and 10 ms at 71 and 52 mV for L3 to L2 and L2 to L3 IPSPs, respectively) (Fig. 6). In six layer 4 interneuron pairs in cat, three pairs (involving five interneurons) yielded IPSPs (Figs 9 and 11
). Each of the three pairs involved interneurons with very different morphologies. The large CCK immunonegative interneuron was presynaptic to one small flask-shaped interneuron in deep layer 4 and postsynaptic to another (Fig. 11
). A bipolar interneuron in upper layer 4 was presynaptic to a small multipolar interneuron also in upper layer 4 (Fig. 9
). In addition, in the only such test performed, an interneuron in upper layer 4 inhibited an interneuron in layer 3. These data suggest that the connectivity between interneurons in all layers and even between adjacent layers and in both species is high (average ratio 1:1.9), as reported previously in immature rat neocortex (Gibson et al., 1999
) and can involve pairs of cells with very different morphologies (Tamás et al., 1998
). Pooling data from all layers for each of the two species, both the EPSPs and the IPSPs recorded in postsynaptic interneurons were briefer than those recorded in postsynaptic excitatory cells (Table 2
), as described previously for EPSPs in adult cat (Tarczy-Hornoch et al., 1998
) and rat (Thomson et al., 1993a
,b
, 1995
) and IPSPs in adult cat (Tamás et al., 1997
) neocortical interneurons.
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Discussion |
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The major differences observed between the two species were differences of scale, with a wider range of neuronal dimensions and morphologies, broader interneuronal axonal and dendritic arbours and more widely separated connected pairs in cat than in rat. These differences in scale may account for some of the differences in connectivity ratios in a slice preparation, since even in the rat neocortex, which contains relatively small neurons, increasing slice thickness from 400 to 500 µm appeared to increase these ratios in our earlier studies. Although cells were typically recorded in the middle 200 µm of the 500 µm slice and very few of the histologically recovered cells had dendrites that had been severed in the preparation of the slices, the axons of many of the pyramidal cells and larger interneurons reached (and presumably had originally extended beyond) the cut surfaces of the slice. It should therefore be remembered that slice preparations lead to an underestimate of connectivity, possibly of the functional strength of connections and of the extent of large axonal abours.
Excitatory Forward Projections
Connections that might be classified as forward projections, e.g. from layer 4 to layer 3 and from layer 3 to layer 5, share some properties and differ from those that might be described as back projections (e.g. from layers 5 to 3 and 3 to 4). Both types of forward projection involve excitatory inputs to both excitatory and inhibitory neurons and in this might appear to be relatively indiscriminate. There is, however, selectivity in the pyramidal cells targeted by these pathways. In layer 5 the forward projections from layer 3 almost exclusively target the larger pyramidal cells in upper layer 5 with apical dendritic tufts in layer 1 (Fig. 1) (Thomson and Bannister, 1998
) and in the present study most of the identified layer 3 pyramidal targets of layer 4 spiny cells were larger pyramidal cells in deep layer 3 (Fig. 3
). In addition, only one cat layer 3 interneuron (of nine interneurons tested with 31 layer 4 cells) demonstrably excited by layer 4 cells was identified in this study (Fig. 6
). This was an unusually large, parvalbumin immuno-positive interneuron which received inputs from three of three layer 4 cells tested. Similarly, only a handful of interneurons in layer 5 demonstrably innervated by layer 3 pyramidal cells have been described previously (Thomson et al., 1996
). Larger samples are required to determine whether the interneuronal targets of these forward projections do indeed comprise specific sub-classes.
Excitatory Back Projections
No EPSPs were elicited in layer 4 excitatory cells by layer 3 pyramidal cells in this (94 tested pairs) or a previous study (Feldmeyer et al., 1999b, 2002
). Similarly, only very rarely was a layer 3 pyramidal cell found to be excited by a layer 5 pyramidal cell (Thomson and Bannister, 1998
; Reyes and Sakmann, 1999
). The few back projections involving excitatory cells that have been recorded (layer 5 to layer 3) involved very small EPSPs and cell pairs separated further in the horizontal plane than is typical for the very tightly focused forward layer 3 to layer 5 projection. Reverberating excitation between layers 3 and 5 and between layers 4 and 3 is therefore extremely unlikely to occur. The possibility of a significant ascending layer 5 to layer 3 or descending layer 3 to layer 4 back projection that involves excitatory cells in distant columns cannot be discounted, but the targets of the long horizontal collaterals of these pyramidal axons have yet to be fully identified.
The scarcity or absence of intra-columnar back projections to excitatory cells does not arise, simply because the potential presynaptic axons do not innervate the region(s) occupied by the postsynaptic dendrites. Layer 5 pyramidal axons project to layer 3 where they innervate interneurons (Dantzker and Callaway, 2000) and the apical dendrites of other layer 5 pyramids (Deuchars et al., 1994
). Similarly, although layer 3 pyramidal axons do not typically ramify in layer 4 and spiny stellate cell dendrites remain largely confined to this layer, the pyramidal cells in layer 4 do send their spiny apical dendrites into layer 3, where they would be readily accessible to layer 3 pyramidal axons (for examples see Figs 3A and 4
). Moreover, the ascending dendrites of upper layer 4 interneurons are commonly targeted by layer 3 pyramidal axons in layer 3. Excitatory back projections therefore exhibit specificity in the targets they select, powerfully exciting interneurons but avoiding excitatory cells. Although the layer 4 interneurons excited by layer 3 pyramidal cells displayed a range of morphological features, all four that were successfully tested for parvalbumin immunoreacitivity were positive and two (one of which was parvalbumin immuno-positive) were seen to make close membrane appositions with very proximal portions of their pyramidal targets, suggesting, perhaps that this back projection favours proximally targeting interneurons.
Inter-laminar Inhibitory Projections
With the exception of dendrite-targeting double bouquet cells, the axonal arbours of layer 3 interneurons were either confined to the superficial layers or included descending projections to layer 5 and, in previous studies, to layer 6 (Tamás et al., 1997, 1998
), with little or no ramification in layer 4. Indeed, no inhibitory connections from layer 3 interneurons to layer 4 excitatory cells were observed in this study (31 tested pairs in cat, 10 in rat), suggesting that the axons of many layer 3 interneurons, like those of layer 3 pyramidal cells, may avoid spiny layer 4 targets. The major difference in the parallel descending excitatory and inhibitory pathways from layer 3 to layer 5 was the relatively narrower arbours of the interneuronal axons of all types in the deep layers. This may not, however, represent selection of a different target population. In both cases, the somata of the layer 5 pyramidal targets of layer 3 axons lie within a very narrow field, since the interneurons often target proximal, while the pyramidal cells target more distal, dendritic sites. These interneurons and those in layer 5 whose axons ramify extensively in layers 5 and 3 could act to coordinate activity in these two layers, relatively independently of layer 4.
The axons and dendrites of some of the layer 4 interneurons studied here were also largely confined to their layer of origin. However, 10 of 27 layer 4 interneurons recovered had axons and dendrites that extended into layer 3. These ascending projections ranged from single axonal and dendritic branches to very wide axonal arbours involving many collaterals that innervated both layers. Some of the interneurons, situated in middle to upper layer 4, that demonstrably inhibited layer 3 cells also received excitatory input from layer 3. They differ significantly from layer 3 interneurons, however, in that the majority of their dendrites are located in a major thalamo-recipient layer. They are well positioned, therefore, to integrate the inputs they receive from layer 4 and layer 3 excitatory cells with direct thalamic input, particularly those that are parvalbumin immuno-positive, parvalbumin-containing interneurons being a major thalamorecipient class of interneurons in layer 4 (Staiger et al., 1996). Indeed, the four layer 4 interneurons innervating both layers and excited by layer 3 pyramidal cells that were so tested were all found to be parvalbumin immuno-positive. The large CCK immuno-negative basket cell with a fast, late spiking, stuttering firing pattern may also have been parvalbumin-containing. By generating IPSPs in cell populations in both layers, these cells could synchronize activity and/or provide a temporal framework within which efficient and meaningful information transfer between the layers could occur.
The long, myelinated, horizontal axon collaterals of some of the larger, upper layer 4 interneurons generated well-separated, tightly focused clusters of boutons hundreds of micrometres from the soma. Some of these may be similar to the large basket cells described previously (Somogyi et al., 1983; Kisvárday, 1992
), which are reported to be densely interconnected (Kisvárday et al., 1993
). Interestingly, the only layer 3 interneuron (of nine tested) in the cat to receive excitatory input from layer 4 had similar long, myelinated, horizontal axon collaterals. These long collaterals could therefore provide patchy inhibition (Kisvárday and Eysel, 1992
) that was controlled by correlated activity in layers 3 and 4 and direct thalamo-cortical input. No equivalently long, patchy horizontal interneuronal projections were found in the rat in this study, but one such layer 4 cell was reported previously (Thomson et al., 1996
).
Time Course of Synaptic Events in Excitatory and Inhibitory Cells
This survey of synaptic connections within and between cortical layers and in two species also allowed the properties of the synaptic events at different synapses to be compared. The properties of any given class of synapse were similar to those described previously for adult rat and were also found to be very similar when similar connections in rat and cat were compared. Synaptic events recorded in inhibitory interneurons were on average much briefer than those recorded in excitatory cells in both species, as reported previously for EPSPs in fast spiking interneurons in rat neocortex that display only a modest N-methyl-D-aspartate receptor-mediated component (Angulo et al., 1999). These brief time courses and the very high connectivity ratios (>1:2) found when pairs of interneurons were tested support the proposed role for interneuronal circuits in the generation/maintenance of the fast gamma rhythms associated with attention and arousal (Buzsáki and Chrobak, 1995
; Traub et al., 1996
). Whether these interneurons are as commonly interconnected via electrical junctions as they are in the developing rat neocortex (Galarreta and Hestrin, 1999
; Gibson et al., 1999
; Támas et al., 2000) was not determined in the present study. No events indicative of such connections were apparent. It is, however, possible that our recording conditions biased existing gap junctions towards the closed state and that under appropriate conditions in vivo, perhaps under the influence of ascending neuro-modulatory systems, these circuits involve both electrical and chemical junctions. The longer duration of IPSPs in pyramidal and spiny stellate cells, in addition to their slower and more powerfully accommodating firing properties, help to explain why networks of excitatory cells do not themselves generate these faster rhythms, firing at best perhaps only on alternate cycles. Their firing is, however, phase locked to these faster oscillations generated by interneuronal circuits. The phase locking will facilitate synchronous activity within selected sub-populations of spiny cells.
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Footnotes |
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This work was supported by the Medical Research Council, Novartis Pharma and the Wellcome Trust. Monoclonal antibody 9303 raised against gastrin/CCK was provided by CURE/Gastroenteric Biology Centre, Antibody/RAI Core, NIH grant DK41301. Polyclonal antibody R301 to parvalbumin was a kind gift from Professor K. Baimbridge, University of British Columbia, Canada.
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