| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1Hertie-Institute for Clinical Brain Research, Department of Cognitive Neurology, University Tübingen, Tübingen; and 2Department of Clinical Neurobiology, University Hospital of Neurology, Heidelberg, Germany
Submitted 4 November 2005; accepted in final form 15 May 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
-aminobutyric acid type B (GABAB) receptors. Blockade of GABAB receptors also abolished the known sublinear addition of inhibitory response duration after repetitive electrical stimulation. Furthermore, evoked inhibition was weaker and longer in connexin 36 knockout (KO) mice that feature decoupled cortical inhibitory networks. In supragranular layers of KO mice even an unusually long excitatory response (
50 ms) appeared that was never observed in wild-type (WT) mice. Furthermore, the spread and duration of very fast oscillations (>200 Hz) evoked by microstimulation at a short latency were strongly enhanced in KO mice. In the spatial domain, lack of connexin 36 unmasked a strong anisotropy of inhibitory spread. Although its reach along layers was almost the same as that in WT mice, the spread across cortical depth was severely hampered. In summary, the present data suggest that connexin 36–coupled networks significantly shape the electrically evoked cortical inhibitory response. Electrical coupling renders evoked cortical inhibition more precise and strong and ensures a uniform spread along the two cardinal axes of neocortical geometry. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Szentágothai 1978). Besides setting the level of depolarization and firing rate of cortical neurons (Hentschke et al. 2005), cortical inhibition plays an important role in shaping temporal (Blatow et al. 2003; Galarreta and Hestrin 2001b; Hormuzdi et al. 2001; Whittington et al. 1995) and spatial properties of cortical activity (Bolz and Gilbert 1986; Moore and Nelson 1998). Most, if not all, cortical inhibitory interneurons use -aminobutyric acid (GABA) as a transmitter and act by ionotropic GABAA receptors with fast and medium time course (Drexler et al. 2005; Fritschy and Brünig 2003), although slower kinetics of synaptic inhibitory responses are known to exist in subcortical structures (Banks et al. 1998; Huntsman et al. 1999). A second type of GABA receptor through which cortical interneurons affect other neurons is the metabotropic GABAB receptor, which shows very slow temporal properties with time constants >100 ms (Connors et al. 1988; Mott and Lewis 1994). The inhibitory network consists of distinct classes of interneurons characterized by histochemical staining, connectivity, expression of receptors, intrinsic membrane characteristics, and dendritic architecture (Kawaguchi and Kondo 2002; Markram et al. 2004). Remarkably, recent studies have indicated that coupling by connexin 36–containing electrical synapses organizes at least some of these interneuron subtypes into distinct syncytia (Beierlein et al. 2000; Blatow et al. 2003; Galarreta and Hestrin 1999; Gibson et al. 1999; Hormuzdi et al. 2001). Electrical coupling within inhibitory networks is important because it allows for tight synchronous firing—not only among the individual members of a syncytium but also of those neurons that communicate with the syncytium by chemical synapses (Connors and Long 2004; Galarreta and Hestrin 2001a). These features occasioned the notion that electrically coupled inhibitory networks may play a decisive role in generating and/or controlling rhythmic activity of the entire neocortical network (Buhl et al. 2003; Whittington et al. 1995).
In this study we investigated how electrical coupling within inhibitory networks affects spatiotemporal activation patterns evoked by intracortical microstimulation. Electrical stimulation has been of general importance for experimental neurobiology (Tehovnik 1996) and has recently gained new momentum in view of its prospective application in neuroprostheses (Nicolelis 2001; Rauschecker and Shannon 2002; Zrenner 2002). Indeed, a large body of work from intracellular and extracellular recordings in the neocortex in vitro and in vivo described a close correlation of both excitatory and inhibitory effects after electrical stimulation. Despite some variation across methods and preparations, the common finding was that excitation is evoked at short latencies and was always followed by a strong and long-lasting inhibitory response (see references in Butovas and Schwarz 2003). Assessing spatial and temporal parameters of the responses as well as electrical parameters of the stimulation indicated that the inhibition was remarkably static in its temporal characteristics and extended 1.35 mm from the stimulation site (Butovas and Schwarz 2003). Its duration was >100 ms and was almost unaffected by 1) changes in near threshold stimulus intensity, 2) the distance to the stimulation site, and 3) double-pulse stimulation within the time span of its duration. The latter feature, in particular, suggested that it is synaptic inhibition that plays a major role in generating the inhibitory response because afterhyperpolarization or synaptic depression, the two major alternatives, would not be compatible with a virtual absence of prolonged inhibition after double-pulse stimulation (Butovas and Schwarz 2003). Its stereotyped and long-lasting characteristics can be well explained by a combination of the highly synchronized action of electrically coupled inhibitory networks and the usage of long-lasting inhibitory synaptic transmission. From the group of GABAergic synaptic mechanisms known to exist in neocortex, GABAB-receptor–mediated inhibitory postsynaptic potentials (IPSPs) would best match the remarkable characteristics of evoked inhibition, that is, the long duration of 200 ms and the resistance to repetitive presynaptic action potentials (Thomson and Destexhe 1999). In the present work we found evidence for these conjectures using a combination of stimulation and multisite recording in genetically modified connexin 36 knockout mice (Hormuzdi et al. 2001) lacking electrical coupling between neocortical interneurons (Deans et al. 2001).
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Nineteen mice (body weight 18.3–28.1 g) of both sexes and both genotypes [ten wild-type (WT) and nine connexin 36–deficient mice (KO) were used in the present study]. The generation of connexin 36 knockout mice is described elsewhere (Hormuzdi et al. 2001). All experimental and surgical procedures were performed in accordance with the policy and the use of animals in neuroscience research of the Society for Neuroscience and German Law. The mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylacine, administered intraperitoneally (ip, 10 mg/kg). The depth of anesthesia was maintained with additional doses of ketamine to keep the hind-limb reflex to a painful stimulus below threshold. The animal's rectal temperature and heart rate were constantly monitored. The temperature was adjusted to 37°C using a controlled heating pad (Fine Science Tools, Heidelberg, Germany). For recordings from neocortex, animals were placed in a stereotaxic apparatus and a craniotomy was performed to expose the primary somatosensory cortex. After removal of the dura mater a multielectrode array, consisting of a row of seven electrodes, was lowered into the neocortex at an angle of 90° with respect to its surface using a hydraulic micropositioner (Kopf 650; David Kopf Instruments, Tujunga, CA). In some experiments we varied stimulation sites using a three-electrode array of individually movable electrodes (EPS, Alpha Omega Engineering, Nazareth, Israel). The middle one recorded from units in the supragranular or infragranular layers; the outer ones, used for stimulation, were moved during the experiment from infra- to supragranular sites. After placement of the electrode array the cortex was covered with mineral oil to prevent drying. The characteristic of recording sites was assessed using either an air puff, originating from a plastic tube (diameter 
0.5 mm) and directed against the whiskers (duration of pulse: 20 ms; distance to whisker pad: 2–3 cm), and/or a cotton swab lightly touching parts of the body. All recordings presented here were performed at sites at which stimulation of the contralateral whisker pad elicited clear unit responses in at least a subset of electrodes. The data of the present study were thus recorded in whisker and neighboring snout representations. Recording sites were marked with electrolytic lesions after the experiment was completed. The animal was deeply anesthetized with barbiturates and perfused through the aorta using phosphate buffer (0.1 M) followed by paraformaldehyde (4% in phosphate buffer). The brain was processed using standard histological procedures. The recording layer was assessed by investigating lesions in Nissl-stained sections oriented orthogonally to the surface of the neocortex.
Electrodes
Multielectrode arrays were custom made in our laboratory. Seven pulled and ground glass–coated platinum tungsten electrodes (shank diameter: 80 µm; diameter of the metal core: 23 µm; free tip length: about 8 µm; impedance: >2 M
; Thomas Recording, Giessen, Germany) were mounted inside a 1 x 7 array of polyimide tubing, for which the distance between tips was set to 300 µm (HV Technologies, Trenton, GA). The free ends of the electrodes were soldered to Teflon-insulated silver wires (Science Products, Hofheim, Germany), which in turn connected to a microplug (Bürklin, Munich, Germany). The electrode at one end of the row was used for electrical stimulation and was placed inside a stainless steel tube that was connected to ground during the recording sessions. The same electrodes were used in the experiment in which three individual electrodes were manipulated.
Electrophysiology
The electrophysiological recordings of extracellular signals were performed using a multichannel extracellular amplifier (MultiChannelSystems, Reutlingen, Germany; gain 5,000; sampling rate 20 kHz; band-pass filter at cutoff frequencies of 200 and 5,000 Hz). Action potentials were extracted from the voltage traces off-line by a threshold and stored as cutouts of 2-ms length on the hard drive of a PC. Spike sorting was performed with PCA clustering using a MATLAB-based (The MathWorks, Natick, MA) program (Egert et al. 2002). Firing rates given in this report always pertain to units recorded with individual electrodes. Through the first electrode in the row of electrodes rectangular biphasic current pulses (cathodal first) were delivered at the rate of 1 Hz using a programmable stimulator (STG 1008; MultiChannelSystems). The electrical stimuli were identical to an earlier study and encompassed the threshold to elicit neuronal responses in rats (Butovas and Schwarz 2003). The stimulus amplitude was fixed to 8 µA and the intensity was changed by varying the pulse duration between 100 and 600 µs in 100-µs steps (charge transfer of 0.8 to 4.8 nC, respectively, varied from pulse to pulse in pseudorandom fashion).
Pharmacology
To investigate effects of GABAB blockade on electrically evoked inhibitory response pattern, CGP 46381 (Tocris Cookson, Ellisville, MO), dissolved in saline (150 mg/kg ip), was administered. The effect of this intervention was monitored by suprathreshold single electrical pulses (charge transfer 4.8 nC) applied at a frequency of 0.2 Hz for 120 min after injection.
Data analysis
Analysis of inhibitory responses was performed as described in a previous report (Butovas and Schwarz 2003; see their Fig. 5D). In short, the spike renewal density function [poststimulus time histogram (PSTH), 1-ms bin width; Abeles 1982] was low-pass filtered by passing a Gaussian (kernel length 50 bins) over it. A unit was classified as responding if the firing rate undershot three quarters of the spontaneous firing (fspont). Duration of the inhibitory response was measured from the stimulation to the point in time where the firing rate crossed this value again. To calculate the response frequency fresp and the strength of the response we took the average frequency within the interval 12 to 80 ms after stimulus onset as fresp. The strength of the inhibitory response was computed as 1 – (fresp/fspont).
|
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). However, there were some quantitative differences with respect to rats. The average duration of the inhibition was significantly shorter (82.5 ± 19.2 ms, n = 132 in mice vs. 118 ± 20.6, n = 186 in rats; Mann–Whitney U test, P < 0.01). In addition, the responsiveness of neurons to local stimulation was lower than that in rats. Although inhibitory response frequency was significantly different from zero at a stimulus intensity of 1.6 nC (as in rats), the 50% mark of response frequency was reached at a stimulus intensity of only 3.2 nC in mice (compare Fig. 5A to Fig. 6 of Butovas and Schwarz 2003). Other features of the inhibitory response, however, closely matched those observed in rats. First, the duration was statistically independent of the distance from the stimulation electrode and the stimulation intensity (two-way ANOVA, P > 0.05, n = 114, Fig. 5C). Second, inhibitory response strength decreased with distance to the stimulation electrode (two-way ANOVA, P < 0.05, n = 114). Third, double-pulse stimulation within the inhibitory period did not elongate the inhibition (Fig. 2A).
|
|
|
|
In principle, diminishing excitatory drive, hyperpolarizing transmembrane currents, and synaptic GABAergic transmission may be at the basis of the inhibitory response (Butovas and Schwarz 2003). Because we were interested in the third possibility—the contribution of the GABAergic system to the firing rate depression—we aimed at showing that it is amenable to the interference with postsynaptic GABA receptors. We chose to block GABAB receptors because, first, our working hypothesis entailed that GABAB receptors may play a role for the mediation of the long-lasting inhibitory response. A second reason was that the blockade of GABAB receptors is readily facilitated by the relatively benign effect of GABAB receptor blockade on gross cortical activity patterns (in sharp contrast to GABAA receptor blockers) and the availability of CGP 46381, a highly specific GABAB receptor blocker that can be administered systemically (Curtis and Lacey 1994; Hershman et al. 1995; Olpe et al. 1993). By using CGP 46381 we consistently found a reduction of the duration of the inhibitory period. In the example shown in Fig. 1 the inhibition was almost completely abolished starting about 30 min after drug application and the spontaneous firing rate increased from 15.3 to 68.4 Hz. This effect was reversed roughly 2 h after drug administration. The reduction in duration of the inhibitory period and the increase in spontaneous firing rate were found in three animals (from 146, 116, and 81 to 71, 43, and 0 ms, respectively; spontaneous firing rate changed from 38, 6.27, and 15.3 to 160, 11.7, and 68.4 Hz, respectively). Stimulating with double pulses showed a sublinear summation of the duration of inhibition in the three animals (Fig. 2A). In two animals that still showed some inhibition at the time of maximal GABAB receptor blockade, the opposite—a supralinear elongation of the remaining inhibitory response—was observed (Fig. 2B). These results strongly indicate a contribution of inhibitory synapses using GABAB receptors to the suppression of firing rate after intracortical stimulation. Furthermore, GABAB receptors seem to play a dominant role in determining the sublinearity of summation of response duration with repetitive stimuli.
Shape of inhibition is dependent on the presence of connexin 36–mediated electrical coupling
If the observed inhibition is at least partly of synaptic origin the question arises with respect to what impact the peculiar organization of inhibitory networks could have on the characteristics of the inhibitory response. Of particular interest is the syncytium-like organization of inhibitory networks by gap junctions containing connexin 36 (Hormuzdi et al. 2001). In studying KO mice, we could readily evoke inhibition. However, several features were distinct from those observed in WT mice (Fig. 3A). First, the duration of the response in KO mice was longer than that observed in WT mice (158.3 [131.4–200.1] ms; n = 171 in KO mice vs. 83.5 [71.0–90.8] n = 170 in WT mice;, Mann–Whitney U test, P < 0.05, Fig. 3B). Second, in some cases an excitatory response lasting for about 50 ms occurred (Fig. 3C), a feature that was never observed in WT mice. Interestingly, the occurrence of this prolonged excitation showed a clear pattern across cortical depth. Recording sites were histologically identified in all nine KO mice. Of the identified recording sites, prolonged excitation was the predominant pattern in supragranular layers (it was absent only in some cases close to the stimulus electrode) but in infragranular layers was missing (with the exception of one case; Fig. 3D).
Next, we asked whether prolonged excitation could be attributable to fast oscillatory activity (FO), previously observed after sensory or electrical surface stimulation (Jones et al. 2000; Staba et al. 2003). Indeed our recordings demonstrated that FOs were evoked by electrical stimulation despite the absence of connexin 36 (Fig. 4) and displayed comparable peaks between 200 and 300 Hz in the power spectrum (data not shown). FOs of comparable amplitudes were never observed to occur spontaneously. In KO mice the FOs appeared extended and their duration was clearly increased. However, the spatial pattern was reversed with respect to that seen for the prolonged excitation. As exemplified in Fig. 4, A–D, FO duration was most conspicuously elongated in infragranular layers and less so in supragranular layers, as would have been expected from the distribution of the prolonged excitation. In infragranular layers the elongation of FOs measured 300 µm away from stimulation electrode at 4.8-nC stimulation intensity was 9.4 ms (KO: 23.1 [20.7–25.6] ms, n = 9, WT: 13.7 [12.7–15.7] ms, n = 12; Mann–Whitney U test, P = 0.000), whereas it reached only 2.4 ms in supragranular layers: KO: 14.9 [12.9–16.0] ms, n = 15, WT: 12.5 [9.9–13.8] ms, n = 13; Mann–Whitney U test, P < 0.05). Furthermore, close inspection of poststimulus voltage traces (Fig. 4E) clearly showed that extended FOs were not accompanied by action potentials. On the other hand, recordings in supragranular layers showed only a small FO content, which occurs before the action potentials that make up the elongated excitation (Fig. 4F). In summary, these results do not argue in favor of FOs being at the basis of the prolonged excitation that precedes the inhibition in supragranular layers of KO mice.
|
We first investigated the spatial distribution of inhibitory responses in WT and KO mice with respect to horizontal cortical distance (Fig. 5, A and B). Surprisingly, the spread of inhibition along layers was very similar in WT and KO mice, although there was a certain tendency for inhibition in KO mice to be spread out more across the cortical surface at medium stimulus intensities (which reached significance at some parameter combinations: the bars with white top in Fig. 5, A and B are statistically different between WT and KO mice, 
2 test, P < 0.05). In particular, near-threshold intensities of 1.6–2.4 nC evoked inhibitory responses at very distant sites (1,800 µm), a feature absent in WT mice. Figure 5, C and D demonstrates that the typical elongation of inhibitory duration was seen irrespective of location and stimulus intensities. The distribution of durations across these parameters was flat in WT mice (two-way ANOVA, P > 0.05, n = 114), whereas in KO mice duration increased with higher charge transfer (two-way ANOVA, P < 0.05, n = 149).
Next, we investigated the spread of inhibition between supra- and infragranular layers through the cortical depth. Experiments in WT mice showed that horizontal spread of inhibition is the same, independent of the location of recording and stimulation electrodes in supra- or infragranular layers. The horizontal connections in supragranular (S-S) and infragranular (I-I) layers displayed inhibitory responses at an intensity close to threshold (7/10 at 3.2 nC) as did the connections that crossed the cortical depth (S-I and I-S; Fig. 6C). In KO mice the duration of inhibition in the horizontal direction was elongated as reported above and the threshold intensities were comparable to those in WT mice (2.4 nC). Upon observing the connections that traversed the cortical depth, however, the picture changed dramatically. No inhibitory response could be observed in the range of stimulus intensities applied (0.8–4.8 nC; S-I, n = 5; I-S, n = 10). Responses could be evoked only by unusually high stimulus intensities. In particular, the transmission S-I was virtually blocked, and in only two of five units shallow and long inhibitory responses at very long latencies about 300 ms were evoked by using exceedingly large stimulus intensities of 64 nC (Fig. 6D, gray PSTH). In I-S orientation 10 of 10 units could be driven by increasing the stimulus intensity to 12 nC (Fig. 6B, gray PSTH). The shape of this inhibitory response was comparable to that obtained with S-S direction (duration: Mann–Whitney U test, P > 0.05). Notably, the presence of elongated excitation depended on the location of the recording site. It was typically present in S-S and I-S situations but not in the other two. Therefore the prolonged excitatory responses revealed by lack of connexin 36 are determined by the recipient neural structures rather than the location of the electrical stimulation.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) is susceptible to pharmacological interference with GABAergic synaptic transmission. Furthermore, the presence of connexin 36, the constituent of gap junctions coupling intracortical inhibitory networks, shapes the spatial spread, strength, and duration of the response in complex ways. Synaptic origin of inhibitory response
Intraperitoneal administration of CGP 46381 at a dose of 150 mg/kg in our study induced an increase of spontaneous firing rate and a reduction of electrically evoked inhibition. These findings can be attributed to the effect of GABAB receptors, given the well-established high specificity of CGP 46381 to GABAB receptors (Brucato et al. 1996; Froestl et al. 1996; Lingenhoehl and Olpe 1993). In particular, Olpe and colleagues (1993) demonstrated high specificity of CGP 46381 to GABAB receptors by showing that concentrations tenfold higher than needed to block late IPSPs were inactive in a battery of receptor assays including GABAA. The dose used in the present study was fivefold higher than the reported threshold dose of 30 mg/kg (Olpe et al. 1993) and thus was well within the range of specific action to GABAB receptors. The pattern of interference—a reduction of evoked inhibition duration combined with an elevation of spontaneous firing—corresponds well to effects of GABAB blockers assessed in vitro by a wealth of studies (e.g., Connors et al. 1988). This close match suggests that receptors and inhibitory circuits located locally in the neocortex very likely contributed to it. Moreover, the short latency and quick succession of fast excitatory and inhibitory responses indicating monosynaptic conveyance (Butovas and Schwarz 2003) also speak in favor of this notion. Notwithstanding these arguments, possible contributions from subcortical GABAB receptors (e.g., those activated by reticular thalamic neurons in the thalamus) to the shortening of the inhibitory response by CGP 46381 cannot be excluded.
In a previous study (Butovas and Schwarz 2003) we proposed that the reduction of spontaneous firing rates elicited by microstimulation is not a result of afterhyperpolarization or decrement of network excitability. The main argument on which this statement was based was that double stimulation did not elongate the inhibitory response, a feature at odds with predictions from the notion that calcium-gated potassium channels (afterhyperpolarization) or synaptic depression is responsible. Our present finding that sublinear summation was abolished by GABAB receptor blockade adds supporting evidence to this view. Sublinear summation fits well with the widely accepted notion that repetitive or cooperative action of many GABAergic interneurons may be required to gate extrasynaptically located GABAB receptors (Isaacson et al. 1993; Mody et al. 1994; Thomson and Destexhe 1999; Thomson et al. 1996). Interestingly, GABAB receptor blockade reversed the summation from sublinear to supralinear summation, rather than simply making it linear. This raises questions about possible enhancement of GABAA-based transmission under blockade of GABAB receptors. One possible mechanism—to be elucidated by future experimentation—is that blockade of presynaptic GABAB receptors leads to an enhancement of GABAergic transmission with accompanying adjustments in the dynamics of excitatory and inhibitory synaptic transmission (Deisz and Prince 1989; Howe et al. 1987).
Temporal coordination of electrically evoked cortical inhibition
We found that the inhibitory response to electrical stimulation in KO mice differed from that in WT mice because it lasted longer and gave rise to excitatory responses of duration 50 ms. This sluggishness of the long inhibitory response in KO mice speaks in favor of a certain weakness and temporal imprecision of the inhibitory response in the absence of connexin 36. It is in line with the view that the presence of gap junctions in cortical inhibitory networks plays a major role in temporally shaping firing patterns (Beierlein et al. 2000; Blatow et al. 2003; Hormuzdi et al. 2001). In view of the suggested contribution of GABAB receptors to the long inhibitory response, it should be recalled that many neurons cooperate in eliciting GABAB-receptor–mediated responses. The need to activate extrasynaptic receptors implies that the temporal precision of such cooperation is determined by the speed of reuptake of GABA from the synaptic cleft, which has been reported to be faster than the time course of a miniature GABAA inhibitory postsynaptic current (Isaacson et al. 1993; Otis and Mody 1992; Solis and Nicoll 1992; Thompson and Gähwiler 1992). Functional gap junctions enable the inhibitory system to achieve high temporal precision in the millisecond (Beierlein et al. 2000; Galarreta and Hestrin 1999), and the lack of synchrony in KO mice (Blatow et al. 2003; Hormuzdi et al. 2001) is predicted to have a significant effect on the efficacy and precision with which GABAB receptors can be recruited. In conclusion, we interpret the sluggishness of the inhibition in KO mice as an expression of the impairment of temporal coordination (and concomitant inefficacy of GABAB receptor activation) within the inhibitory network when electrically decoupled.
Spatial coordination of electrically evoked cortical inhibition
We found that the horizontal spread of inhibition was principally intact in KO mice but its transmission across cortical depth required connexin 36–containing electrical synapses. In WT mice, a stimulus intensity of 2.4–3.2 nC caused an inhibitory response that spread to the neighboring column. In the absence of connexin 36, inhibitory activity evoked by the same stimulus intensity was unable to straddle the granular layer and reached the neighboring column only at the same depth as the stimulation site. In principle, the diminished cross-layer inhibitory responses in the absence of connexin 36 may reflect properties of either inhibitory or excitatory axons straddling the granular layer (the latter giving rise to a polysynaptic response by local inhibitory neurons). It is a well-established fact that intrinsic cortical connections are highly specific (Kawaguchi and Kubota 1997): the likelihood of finding a connection between a given pair of neurons depends strongly on the location of the cells (i.e., layer) and their neuronal type (i.e., spiny, smooth, excitatory, inhibitory, etc.). Indeed, some known characteristics of specific connections, reported in visual cortex, tentatively match the present finding of weaker transmission of inhibition across the granular layer compared with horizontal directions and the asymmetry of I-S versus S-I signal transfer, and therefore may guide future attempts to elucidate the underlying mechanisms: Inhibitory axons generally show little tendency to straddle the granular layer in both directions (Watts and Thomson 2005). Furthermore, in the S-I direction, which is virtually blocked in the absence of connexin 36, layer 3 pyramids in visual cortex show a prominent excitatory connection to large layer 5 pyramidal neurons, but they do not target interneurons there. In the reverse direction (I-S, which shows existent but severed inhibitory transmission in KO mice), layer 5 pyramids do not target layer 3 pyramids, although connections to inhibitory interneurons in that layer have been found (Watts and Thomson 2005). In the horizontal direction, direct and indirect transmissions of inhibitory responses along layers have been well documented. Inhibitory basket cells extend axons as far as 1 mm in the horizontal direction (Kisvárday 1992) and about one fifth of the synaptic boutons on long horizontal axons of pyramidal cells have been reported to target putative inhibitory neurons (McGuire et al. 1991).
In line with our finding of unimpaired horizontal spread, synchronous oscillations of the membrane potential at 4 Hz (at which coupled pairs show a high coupling ratio (Galarreta and Hestrin 1999) fall off significantly within a horizontal distance of 600 µm within layer 4 (Beierlein et al. 2000)—a distance that is far smaller than the spatial spread of the inhibitory response in supra- and infragranular layers observed in the present study. Thus conductance through gap junctions between inhibitory interneurons in horizontal direction may be optimized to allow for temporal coordination but does not contribute much to the spatial spread of activity. However, the present finding of impaired spatial spread across cortical depth in KO mice does not fit this picture. Such an anisotropic pattern was not predicted from the fairly homogeneous distribution of connexin 36 across cortical layers (Belluardo et al. 2000). A possible explanation is that coupling ratios across layers are higher than those assessed in horizontal direction—a possibility that needs to be tested by future experiments using double recordings in vitro.
Further layer-specific characteristics of inhibition were unmasked by the absence of connexin 36 in KO mice. The prolonged elevation of firing rate was seen exclusively in supragranular layers and FOs were particularly affected in infragranular layers. These findings are in line with previous evidence that morphological and electrophysiological properties of the inhibitory system vary systematically between infra- and supragranular layers. Besides the above-mentioned layer-specific projection patterns, there is evidence that the impact of inhibition differs between infra- and supragranular layers: Counts of GABAergic neurons and terminals per cell have been found to be maximal in layer V in gerbil auditory cortex (Foeller et al. 2001), and the horizontal reach of inhibitory projections, as measured with electrical stimulation in rat somatosensory cortex, has been found to be maximal in infragranular layers (Salin and Prince 1996a). Furthermore, the frequency of spontaneous inhibitory input differs between supra- and infragranular layers (Jones and Woodhall 2005; Salin and Prince 1996b).
It is clear that more quantitative data about intracortical distribution of projections and their targets, as well as variation of electrical and pharmacological properties of inhibitory circuits, are needed, before the depth-specific effects observed in the present study can be fully understood. Furthermore, it must be borne in mind that the interpretation of our data with respect to cortical microcircuits rests on the assumption that the specificity of intrinsic connections in KO mice matches that in WT mice. Conversely, and most important, our findings highlight the predominance of gap junctions for shaping inhibitory responses to electrical stimulation in the neocortex: Electrical signaling is a decisive factor in establishing the cohesion of inhibitory response characteristics across cortical layers. The spread through cortical layers and both the timing and the precision of the inhibitory response depend on the presence of connexin 36.
|
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: C. Schwarz, Hertie-Institute for Clinical Brain Research, Department of Cognitive Neurology, University Tübingen, Otfried Müller Str. 27, 72076 Tübingen, Germany (E-mail: cornelius.schwarz{at}uni-tuebingen.de)
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Banks MI, Li TB, and Pearce RA. The synaptic basis of GABAA,slow. J Neurosci 18: 1305–1317, 1998.
Beierlein M, Gibson JR, and Connors BW. A network of electrically coupled interneurons drives synchronized inhibition in neocortex. Nat Neurosci 3: 904–910, 2000.[CrossRef][ISI][Medline]
Belluardo N, Mudó G, Trovato-Salinaro A, Le Gurun S, Charollais A, Serre-Beinier V, Amato G, Haefliger JA, Meda P, and Condorelli DF. Expression of connexin36 in the adult and developing rat brain. Brain Res 865: 121–138, 2000.[CrossRef][ISI][Medline]
Blatow M, Rozov A, Katona I, Hormuzdi SG, Meyer AH, Whittington MA, Caputi A, and Monyer H. A novel network of multipolar bursting interneurons generates theta frequency oscillations in neocortex. Neuron 38: 805–817, 2003.[CrossRef][ISI][Medline]
Bolz J and Gilbert CD. Generation of end-inhibition in the visual cortex via interlaminar connections. Nature 320: 362–365, 1986.[CrossRef][Medline]
Brucato FH, Levin ED, Mott DD, Lewis DV, Wilson WA, and Swartzwelder HS. Hippocampal long-term potentiation and spatial learning in the rat: effects of GABAB receptor blockade. Neuroscience 74: 331–339, 1996.[CrossRef][ISI][Medline]
Buhl DL, Harris KD, Hormuzdi SG, Monyer H, and Buzsáki G. Selective impairment of hippocampal gamma oscillations in connexin-36 knock-out mouse in vivo. J Neurosci 23: 1013–1018, 2003.
Butovas S and Schwarz C. Spatiotemporal effects of microstimulation in rat neocortex: a parametric study using multielectrode recordings. J Neurophysiol 90: 3024–3039, 2003.
Connors BW and Long MA. Electrical synapses in the mammalian brain. Annu Rev Neurosci 27: 393–418, 2004.[CrossRef][ISI][Medline]
Connors BW, Malenka RC, and Silva LR. Two inhibitory postsynaptic potentials, and GABAA and GABAB receptor-mediated responses in neocortex of rat and cat. J Physiol 406: 443–468, 1988.
Curtis DR and Lacey G. GABA-B receptor-mediated spinal inhibition. Neuroreport 5: 540–542, 1994.[ISI][Medline]
Deans MR, Gibson JR, Sellitto C, Connors BW, and Paul DL. Synchronous activity of inhibitory networks in neocortex requires electrical synapses containing connexin36. Neuron 31: 477–485, 2001.[CrossRef][ISI][Medline]
Deisz RA and Prince DA. Frequency-dependent depression of inhibition in guinea pig neocortex in vitro by GABAB receptor feed-back on GABA release. J Physiol 412: 513–542, 1989.
Drexler B, Roether CL, Jurd R, Rudolph U, and Antkowiak B. Opposing actions of etomidate on cortical theta oscillations are mediated by different gamma-aminobutyric acid type A receptor subtypes. Anesthesiology 102: 346–352, 2005.[CrossRef][ISI][Medline]
Egert U, Knott T, Schwarz C, Nawrot M, Brandt A, Rotter S, and Diesmann M. MEA-Tools: an open source toolbox for the analysis of multi-electrode data with MATLAB. J Neurosci Methods. 117:33–42, 2002.[CrossRef][ISI][Medline]
Foeller E, Vater M, and Kossl M. Laminar analysis of inhibition in the gerbil primary auditory cortex. J Assoc Res Otolaryngol 2: 279–296, 2001.[Medline]
Fritschy JM and Brünig I. Formation and plasticity of GABAergic synapses: physiological mechanisms and pathophysiological implications. Pharmacol Ther 98: 299–323, 2003.[CrossRef][ISI][Medline]
Froestl W, Mickel SJ, Von Sprecher G, Bittiger H, and Olpe HR. Pharmacol Commun 2: 52–56, 1992.
Galarreta M and Hestrin S. A network of fast-spiking cells in the neocortex connected by electrical synapses. Nature 402: 72–75, 1999.[CrossRef][Medline]
Galarreta M and Hestrin S. Electrical synapses between GABA-releasing interneurons. Nat Rev Neurosci 2: 425–433, 2001a.[CrossRef][ISI][Medline]
Galarreta M and Hestrin S. Spike transmission and synchrony detection in networks of GABAergic interneurons. Science 292: 2295–2299, 2001b.
Gibson JR, Beierlein M, and Connors BW. Two networks of electrically coupled inhibitory neurons in neocortex. Nature 402: 75–79, 1999.[CrossRef][Medline]
Hentschke H, Schwarz C, and Antkowiak B. Neocortex is the major target of sedative concentrations of volatile anaesthetics: strong depression of firing rates and increase of GABA receptor-mediated inhibition. Eur J Neurosci 21: 93–102, 2005.[CrossRef][ISI][Medline]
Hershman KM, Freedman R, and Bickford PC. GABAB antagonists diminish the inhibitory gating of auditory response in the rat hippocampus. Neurosci Lett 190: 133–136, 1995.[CrossRef][ISI][Medline]
Hormuzdi SG, Pais I, LeBeau FE, Towers SK, Rozov A, Buhl EH, Whittington MA, and Monyer H. Impaired electrical signaling disrupts gamma frequency oscillations in connexin 36-deficient mice. Neuron 31: 487–495, 2001.[CrossRef][ISI][Medline]
Howe JR, Sutor B, and Zieglgansberger W. Baclofen reduces post-synaptic potentials of rat cortical neurones by an action other than its hyperpolarizing action. J Physiol 384: 539–569, 1987.
Huntsman MM, Porcello DM, Homanics GE, DeLorey TM, and Huguenard JR. Reciprocal inhibitory connections and network synchrony in the mammalian thalamus. Science 283: 541–543, 1999.
Isaacson JS, Solis JM, and Nicoll RA. Local and diffuse synaptic actions of GABA in the hippocampus. Neuron 10: 165–175, 1993.[CrossRef][ISI][Medline]
Jones MS, Macdonald KD, Choi B, Dudek FE, and Barth DS. Intracellular correlates of fast (>200 Hz) electrical oscillations in rat somatosensory cortex. J Neurophysiol 84: 1505–1518, 2000.
Jones RS and Woodhall GL. Background synaptic activity in rat entorhinal cortical neurons: differential control of transmitter release by presynaptic receptors. J Physiol 562: 107–120, 2005.
Kawaguchi Y and Kondo S. Parvalbumin, somatostatin and cholecystokinin as chemical markers for specific GABAergic interneuron types in the rat frontal cortex. J Neurocytol 31: 277–287, 2002.[CrossRef][ISI][Medline]
Kawaguchi Y and Kubota Y. GABAergic cell subtypes and their synaptic connections in rat frontal cortex. Cereb Cortex 7: 476–486, 1997.
Kisvárday ZF. GABAergic networks of basket cells in the visual cortex. In: Progress in Brain Research: Mechanisms of GABA in the Visual System, edited by Mize RR, Marc RE, and Sillito AM. Amsterdam: Elsevier Science, 1992, vol. 90, p. 385–405.
Lingenhoehl K and Olpe HR. Pharmacol Commun 3: 49–54, 1993.
Markram H, Toledo-Rodriguez M, Wang Y, Gupta A, Silberberg G, and Wu C. Interneurons of the neocortical inhibitory system. Nat Rev Neurosci 5: 793–807, 2004.[CrossRef][ISI][Medline]
McGuire BA, Gilbert CD, Rivlin PK, and Wiesel TN. Targets of horizontal connections in macaque primary visual cortex. J Comp Neurol 305: 370–392, 1991.[CrossRef][ISI][Medline]
Mody I, De Koninck Y, Otis TS, and Soltesz I. Bridging the cleft at GABA synapses in the brain. Trends Neurosci 17: 517–525, 1994.[CrossRef][ISI][Medline]
Moore CI and Nelson SB. Spatio-temporal subthreshold receptive fields in the vibrissa representation of rat primary somatosensory cortex. J Neurophysiol 80: 2882–2892, 1998.
Mott DD and Lewis DV. The pharmacology and function of central GABAB receptors. Int Rev Neurobiol 36: 97–223, 1994.[ISI][Medline]
Nicolelis MA. Actions from thoughts. Nature Suppl 409: 403–407, 2001.[CrossRef][Medline]
Olpe HR, Steinmann MW, Ferrat T, Pozza MF, Greiner K, Brugger F, Froestl W, Mickel SJ, and Bittiger H. The actions of orally active GABAB receptor antagonists on GABAergic transmission in vivo and in vitro. Eur J Pharmacol 233: 179–186, 1993.[CrossRef][ISI][Medline]
Otis TS and Mody I. Modulation of decay kinetics and frequency of GABAA receptor-mediated spontaneous inhibitory postsynaptic currents in hippocampal neurons. Neuroscience 49: 13–32, 1992.[CrossRef][ISI][Medline]
Peters A. Synaptic specifity in the cerebral cortex. In: Synaptic Function, edited by Edelman GM, Gall WE, and Cowan WM. New York: Wiley, 1987, p. 373–397.
Rauschecker JP and Shannon RV. Sending sound to the brain. Science 295: 1025–1029, 2002.
Salin PA and Prince DA. Electrophysiological mapping of GABAA receptor-mediated inhibition in adult rat somatosensory cortex. J Neurophysiol 75: 1589–1600, 1996a.
Salin PA and Prince DA. Spontaneous GABAA receptor-mediated inhibitory currents in adult rat somatosensory cortex. J Neurophysiol 75: 1573–1588, 1996b.
Solis JM and Nicoll RA. Pharmacological characterization of GABAB-mediated responses in the CA1 region of the rat hippocampal slice. J Neurosci 12: 3466–3472, 1992.[Abstract]
Staba RJ, Brett-Green B, Paulsen M, and Barth DS. Effects of ventrobasal lesion and cortical cooling on fast oscillations (>200 Hz) in rat somatosensory cortex. J Neurophysiol 89: 2380–2388, 2003.
Szentágothai J. The neuron network of the cerebral cortex: a functional interpretation. Proc R Soc Lond B Biol Sci 201: 219–248, 1978.[Medline]
Tehovnik EJ. Electrical stimulation of neural tissue to evoke behavioral responses. J Neurosci Methods 65: 1–17, 1996.[CrossRef][ISI][Medline]
Thompson SM and Gähwiler BH. Effects of the GABA uptake inhibitor tiagabine on inhibitory synaptic potentials in rat hippocampal slice cultures. J Neurophysiol 67: 1698–1701, 1992.
Thomson AM and Destexhe A. Dual intracellular recordings and computational models of slow inhibitory postsynaptic potentials in rat neocortical and hippocampal slices. Neuroscience 92: 1193–1215, 1999.[CrossRef][ISI][Medline]
Thomson AM, West DC, Hahn J, and Deuchars J. Single axon IPSPs elicited in pyramidal cells by three classes of interneurons in slices of rat neocortex. J Physiol 496: 81–102, 1996.
Watts J and Thomson AM. Excitatory and inhibitory connections show selectivity in the neocortex. J Physiol 562: 89–97, 2005.
Whittington MA, Traub RD, and Jefferys JG. Synchronized oscillations in interneuron networks driven by metabotropic glutamate receptor activation. Nature 373: 612–615, 1995.[CrossRef][Medline]
Zrenner E. Will retinal implants restore vision? Science 295: 1022–1025, 2002.
This article has been cited by other articles:
|
|
 |
|
  D. L. Kimmel and T. Moore Temporal Patterning of Saccadic Eye Movement Signals J. Neurosci., July 18, 2007; 27(29): 7619 - 7630. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
