Myelination Defects and Neuronal Hyperexcitability in the Neocortex of Connexin 32-deficient Mice

Bernd Sutor, Cordula Schmolke1,2, Barbara Teubner3, Clemens Schirmer and Klaus Willecke3

Institute of Physiology, University of Munich, Pettenkoferstrasse 12, D-80336 Munich, , 1 Institute of Anatomy, University of Bonn, Nussallee 10, D-53115 Bonn and , 3 Institute of Genetics, University of Bonn, Römerstrasse 164, D-53117 Bonn, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Morphological and electrophysiological studies were performed on neocortices of adult Connexin 32 (Cx32)-deficient mice and wild-type mice to investigate the consequences of a lack of the gap junction subunit Cx32 on neocortical structure and function. Morphometrical analysis revealed a reduced volume fraction of myelin within the neuropil and a decreased thickness of the axonal myelin sheaths in the neocortex of Cx32-deficient mice. Intracellular recordings from neurons in neocortical slice preparations provided evidence for an increased membrane input resistance in neurons of Cx32-null mutant mice as compared to neurons of wild-type mice. Consequently, neurons of Cx32-deficient mice displayed an enhanced intrinsic excitability. In addition, ~50% of the neurons investigated in slices of Cx32-deficient mice responded to afferent stimulation with delayed and large glutamatergic excitatory postsynaptic potentials resembling paroxysmal depolarizations. GABAergic inhibition sufficient to efficiently control synaptic excitability was virtually absent in these cells. The changes in intrinsic membrane properties observed in neurons of Cx32-null mutant mice were independent of the alterations in synaptic function, since increased membrane resistances were observed also in neurons with normal synaptic response pattern. Thus, in the neocortex, lack of Cx32 correlates with myelination defects, alterations in intrinsic membrane properties and dysfunction of inhibitory synaptic transmission.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
In the peripheral nervous system (PNS), the gap junction subunit protein Connexin 32 (Cx32) is an essential constituent of Schwann cells (Bergoffen et al., 1993Go; Scherer et al., 1995Go; Spray and Dermietzel, 1995Go). These cells are required for myelination of peripheral axons. A lack of Cx32 in Schwann cells, as in Cx32-deficient mice (Nelles et al., 1996Go; Anzini et al., 1997Go), leads to changes in the myelination of peripheral nerves associated with a reduction in their conduction velocities (Anzini et al., 1997Go; Scherer et al., 1998Go). Similar disturbances occur in humans suffering from hereditary X-linked Charcot–Marie– Tooth (CMTX) disease, which is characterized by a reduced function of Cx32-gap junction channels in Schwann cells (Bergoffen et al., 1993Go; Nicholson and Nash, 1993Go; Spray and Dermietzel, 1995Go; Ionasescu et al., 1996Go).

In the central nervous system (CNS), Cx32 has been localized predominantly in oligodendrocytes, but also in some types of neurons (Dermietzel et al., 1989Go; Kunzelmann et al., 1997Go; Li et al., 1997Go). Little is known, however, about the function of Cx32 in the CNS. Oligodendrocyte-dependent myelination of CNS axons was found to be unaffected in Cx32-deficient mice (Scherer et al., 1998Go). Furthermore, it is not clear whether, and to what extent, CMTX patients display CNS-related abnormalities. It has been reported that these patients do not develop CNS symptoms (Dyck et al., 1993Go). Another study, however, revealed a reduction in conduction velocity of central nerves of CMTX patients (Nicholson and Corbett, 1996Go).

In rodents, high rates of gap junction-mediated coupling among astrocytes has been found to exist in the immature as well as in the mature neocortex (Gutnick et al., 1981Go; Connors et al., 1984Go). The gap junctions between these glial cells are formed predominantly by Cx43 (Yamamoto et al., 1990Go). The incidence of gap junction-mediated coupling among neocortical pyramidal cells depends on the developmental age. It is prevalent during pre- and early postnatal stages of neocortical development, but is virtually absent in the mature neocortex (Rörig and Sutor, 1996Go). There is little information about gap junction-mediated coupling among neocortical interneurons. Recently, networks of GABA- ergic interneurons connected via gap junctions have been described in the neocortex of young rats (Galarreta and Hestrin, 1999Go, Gibson et al., 1999Go).

From the developmental time course of Cx32 expression deduced from immunohistochemical investigations and mRNA- expression (Dermietzel et al., 1989Go; Nadarajah et al., 1997Go; Prime et al., 1998Go), it seems to be unlikely that Cx32 contributes significantly to the extensive coupling of pyramidal cells observed during the early postnatal periods of neocortical development. The time course of Cx32 expression within the neocortex coincides with that of oligodendrocyte maturation (Parnavelas et al., 1983Go). Therefore, since Cx32 is predominantly expressed in oligodendrocytes, one might primarily expect that this gap junction protein is functionally associated with myelination of neocortical axons. On the other hand, there is evidence for the expression of Cx32 in CNS neurons (Dermietzel et al., 1989Go, Kunzelmann et al., 1997Go, Li et al., 1997Go). Thus, an involvement of Cx32 in the determination of electrophysiological properties of neocortical neurons, in particular of mature neurons, cannot be excluded.

In order to learn more about the function of Cx32 within the neocortex, we performed morphological and electrophysiological studies on neocortices of adult Cx32-deficient mice. We investigated whether a lack of Cx32 caused alterations in basic morphological features of the mouse neocortex and/or changes in the electrophysiological properties of neocortical neurons. Parts of these studies have been published in abstract form (Sutor et al., 1997Go).


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
The generation and characterization of Cx32-deficient mice has previously been described (Nelles et al., 1996Go). The mice of mixed genetic background (C57BL/6 and 129 Sv) were generated and genotyped at the Institute of Genetics, University of Bonn, and sent to the Institute of Physiology, University of Munich, and to the Institute of Anatomy, University of Bonn, for electrophysiological and morphological investigations respectively. As controls, animals of the F1 generation of the inbred strains C57BL/6 and 129 Sv were used. The Cx32 gene has been assigned to the mouse X chromosome (Schwarz et al., 1992Go). Thus, the correct designation of the null mutant genotypes are Cx32–/– or gjb1–/– for females and Cx32–/y or gjb1–/y for males. In the present paper, Cx32-deficient mice were designated Cx32–/–, independent of the sex.

Morphological Studies

Four Cx32-deficient mice and four wild-type mice matched with respect to sex and age [>6 months old (Anzini et al., 1997Go)] were used for morphological studies. After determination of their body weights, the animals were anesthetized (pentobarbitone sodium, 2 ml i.p., 4.4 g/kg) and transcardially perfused. Perfusion was started with 10 ml of solution A consisting of 1% (v/v) glutaraldehyde and 1% (v/v) paraformaldehyde in 0.12 M phosphate buffer containing 0.02 mM CaCl2 (pH 7.4, at room temperature), and followed by 50 ml of solution B comprising 3% glutaraldehyde and 3% paraformaldehyde in the same buffer (Schmolke, 1987Go). Three hours later, the brains were dissected from the skulls and brain stems as well as cerebelli were removed. After determination of weight, the brains were divided into their hemispheres and small blocks containing the parietal region of the cerebral cortex were prepared from each hemisphere. The blocks were immersed for 2 h in solution B and washed overnight with several changes of 0.12 M phosphate buffer (pH 7.4) containing 8% (w/v) dextrose and 0.02 mM CaCl2. The next day, the specimen were postfixed for 4 h with 2% (w/v) OsO4 in 0.12 M phosphate buffer (pH 7.4) containing 7% (w/v) dextrose and 0.02 mM CaCl2, dehydrated in graded ethanol solutions and embedded using propylene oxide in araldite (Durcupan ACM®, Fluka, Buchs, Switzerland).

For light microscopical analysis, semithin sections (1 µm) were prepared. Blocks of the right hemispheres were cut perpendicularly to the cortical surface. From the left hemispheres, serial sections were cut at a tangential plane starting from the pial surface throughout the entire cortex and into the white matter. All sections were stained with a mixture of toluidine blue and pyronin G following a previously published method (Ito and Winchester, 1963Go). For electron microscopical analysis, ultrathin sections were cut from those tissue blocks which were obtained by cutting perpendicularly to the cortical surface. These sections were stained with uranyl acetate and lead citrate and investigated using a Philips 301 electron microscope (Philips, Eindhoven, The Netherlands).

Morphometrical measurements were performed using a semi- automatic image-analyzing system (VIDS V, AMS, Cambridge, UK). Sections from three animals of each experimental group were chosen according to the quality of fixation, in particular with respect to the fine structure of myelin sheaths. Cortical thickness was light microscopically determined from one semithin section of each animal. For this analysis, sections were selected which were cut perpendicular to the cortical surface as perfect as possible. On each section, ten measurements were performed at different sites in the medio-lateral extension of the somatosensory cortex.

In order to evaluate the degree of myelination, the number of myelinated axons per unit area of neuropil, the diameters of the axons, the thickness of their myelin sheaths and the volume fraction of myelin within the neuropil were determined using electron microscopy. From each animal, ten electron micrographs (primary magnification x9100) were taken from lamina VI in sections cut perpendicularly to the cortical surface. The morphometrical measurements were carried out directly on transilluminated negatives of the electron micrographs.

To determine the number of axons per unit area of neuropil, axons were counted in a total area of 420 µm2 on five micrographs of each mouse. For measurement of axonal diameter and thickness of the myelin sheath, the contours of 100 nerve fibers (i.e. axon plus myelin sheath, and the respective axon without myelin sheath) were digitized (Fig. 1Go). Most axons were cut obliquely resulting in ovoidly shaped profiles (see Fig. 3c,dGo). To standardize the measurements, the shortest diameter through the center of gravity of the profile was determined and considered as a measure for the diameter of axons Da(i) and of the nerve fibers Dn(i) respectively (see Fig. 1Go). The thickness of the myelin sheath Dm (i) was calculated according to the formula: Dm (i) = (Dn (i)Da (i))/2.



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Figure 1. Schematic drawing of the myelin sheath of a cross sectioned axon (A) with two different artifacts (arrows) frequently observed. At the inner side of the myelin sheath, some of the lamellae occasionally separate from the sheath and indent into the region that is normally occupied by the axon (two-tailed arrow). At the outer side, lamellae may separate (solid arrow) and protude into the surrounding neuropil (N). The white dotted line indicates the contour of the axon, the dashed–dotted line designates the contour of the nerve fiber. For morphometric analysis, the contours were digitized according to these lines. The artifacts were corrected for by calculating the shortest diameter of the nerve fiber (indicated by the horizontal line, center of gravity is marked by a white diamond). These values were taken as equivalent values for the thickness of the fiber.

 


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Figure 3. Morphology of myelinated axons in the somatosensory cortex of wild-type mice (a,c,e) and Cx32-deficient mice (b,d,f). (a,b) Light microscopical appearance of the deep cortical lamina VI of a wild-type animal (a) and of a Cx32-deficient mouse (b). Semithin sections cut perpendicular to the pial surface. Magnification: x900. (c,d) Ultrastructure of the myelinated axons in deep lamina VI of a wild-type mouse (c) and a Cx32-deficient animal (d). Comparison of the fibers marked with stars in both micrographs shows that the thickness of the myelin sheath (in relation to the axon diameter) is reduced in Cx32-null mutant mice. The arrowheads in (c) indicate artifacts as described in Figure 1Go. Magnification: x22 000. (e,f): High-power electron micrographs of nodes of Ranvier of vertically oriented myelinated axons (A) at the level of lamina IV: (e) wild-type; (f) Cx32-deficient. Note the irregularly shaped and elongated lateral loops in (f). The star in (f) indicates an asymmetrical protrusion of the myelin sheath in the region of the paranodal zone. AG: astroglial process. Magnification: x60 000. The insets in (e) and (f) depict the compact structure of the myelin sheath at higher magnification (xx158 000). In the Cx32-deficient mouse (f), the distance between two major dense lines was larger than in wild-type animals (e).

 
In some sections, the myelin sheaths of axons displayed artifacts caused by imperfect fixation (Hirano and Llena, 1995Go). Myelin sheaths affected by insufficient fixation showed segments with lamellae separated from each other. Some lamellae near the inner surface of the myelin sheath became indented into the axon, thereby altering the shape of the axonal profile. Others appeared protruded from the outer surface of the nerve fiber causing deviations of the contours of the respective nerve fiber (see Fig. 3cGo). To avoid false measurements in these cases, deviations of the contours of axons and nerve fibers caused by fixation artifacts were corrected for during digitization as shown in Figure 1Go.

The volume fraction of myelin was determined by the so-called point-counting method (Weibel, 1979Go). Five electron micrographs of each animal were analyzed by randomly assigning 374 test points to each micrograph (i.e. total number of test points in the neuropil, Pn) and counting the number of test points lying on myelin sheaths (Pm). The volume fraction of myelin Vm was then calculated using the equation: Vm = (Pm/Pn) x 100 (in %).

All quantitative parameters are indicated as mean ± SD. Statistical comparisons between Cx32-deficient and wild-type mice were performed using the unpaired two-tailed t-test (significance level: P < 0.05).

Electrophysiological Studies

Intracellular recordings were performed from neurons in brain slices prepared from the neocortices of 13 adult Cx32-deficient mice and 13 adult control mice of both sexes. The preparation of slices was carried out using the same technique as described previously for rat neocortical slices (Rörig et al., 1995Go, 1996Go). In each experiment, one mouse was deeply anesthetized with isoflurane (Forene®, Abbott, Wiesbaden, Germany) and decapitated. Then, the brain was removed from the skull and incubated for ~1 min in ice-cold and oxygenated artificial cerebrospinal fluid (ACSF). Following removal of the cerebellum and separation of the hemispheres, coronal slices (thickness: 400 µm) of the whole hemisphere were cut using a vibratome (VT 1000E, Leica Instruments, Nussloch, Germany). Slices corresponding to the region of the somatosensory cortex were selected and stored in ACSF at room temperature (18–20°C) for at least 1 h. Individual slices were transferred to a recording chamber, fixed between two nylon meshes and continuously perfused with ACSF at a flow rate of 4–5 ml/min. The ACSF consisted of (in mM): 125 NaCl, 3 KCl, 2.5 CaCl2, 1.3 MgCl2, 1.25 NaH2PO4, 25 NaHCO3 and 10 D-glucose. The solution was saturated with 95% O2 and 5% CO2 in order to maintain a pH value of 7.4 at a recording temperature of 32°C.

Intracellular recordings were performed using glass microelectrodes filled with 2 M potassium acetate containing 0.3 M potassium chloride (electrode resistance: 80–140 M{Omega}) or with 3 M KCl (80–120 M{Omega}). Electrodes were pulled from borosilicate glass capillaries (o.d. 1.5 mm, i.d. 0.86 mm, Clark Electromedical Instruments, Reading, UK). Signals were recorded and amplified by means of a single-electrode current- and voltage-clamp amplifier (SEC-10L, npi, Tamm, Germany) operated in bridge mode. Data were aquired on-line using a CED 1401 interface (Cambridge Electronic Devices, Cambridge, UK) in conjunction with a laboratory computer. The analysis of the recorded data was performed off-line by means of the AutesW software (H. Zucker, Max-Planck-Institute of Psychiatry, Planegg-Martinsried, Germany).

Postsynaptic potentials were evoked by electrical stimulation using a bipolar concentric metal electrode (diam. 100 µm). This electrode was positioned into the white matter underlying the neocortex. Stimulation pulses had a duration of 100 µs and were applied at a frequency of 0.05 Hz. The amplitudes of the stimuli were adjusted by means of an isolation unit.

Drugs were applied by addition to the bathing solution [bicuculline methiodide (Sigma, Deisenhofen, Germany), D-2-amino-5-phospho- novaleric acid (D-2-APV, Sigma) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, Biotrend, Köln, Germany)]. Values are given as mean ± SD. In the case of a normal distribution of data, statistical comparison of two mean values was performed using the unpaired two-tailed t-test, otherwise the non-parametric Mann–Whitney test was applied.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
Morphological Studies

Similar to other investigators (Nelles et al., 1996Go), we observed significant reductions of both body weight and brain weight in Cx32-deficient mice as compared to wild-type mice (Table 1Go). The ratio of body weight to brain weight was not significantly different.


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Table 1 Comparison of morphological parameters determined in wild-type and Cx32-deficient mice
 
On the light microscopical level, the structure of the somatosensory cortex of Cx32-deficient mice was similar to that of wild-type animals (Fig. 2adGo). In Cx32-null mutant mice, the thickness of the neocortex tended to be slightly smaller as compared to that of wild-type animals (Table 1Go). However, the difference was statistically not significant. Similarly, the cytoarchitectonic layering of the somatosensory cortex of Cx32-deficient mice (Fig. 2bGo) displayed a tendency to differ from that of wild-type animals (Fig. 2aGo). In some cases, the widths of laminae V and VI were found to be decreased by 20–25% (compare Fig. 2a and bGo). However, this was not a consistent observation and quantitative analysis of the layer widths resulted in statistically insignificant differences.



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Figure 2. General features of the architecture of the neocortex of wild type-mice (a,c) and of Cx32-deficient mice (b,d,e). (a,b) Semithin coronal sections displaying the entire width of the somatosensory cortex of a wild-type mouse (a) and of a Cx32-null mutant mouse (b). The cytoarchitectonic laminae are indicated by Roman numbers. Vertical bundles of apical dendrites of pyramidal cells are indicated by arrowheads (lamina II/III and lamina IV). Magnification: x160. (c,d) Semithin sections of the somatosensory cortex cut tangentially to the pial surface at the level of lamina IV: (c) wild-type mouse, (d) Cx32-deficient mouse. Cross-sectioned dendritic bundles are indicated by arrowheads. Magnification: x690. (e) Electron micrograph of a dendritic bundle in lamina IV of the somatosensory cortex of a Cx32-deficient mouse. Ultrathin section cut perpendicularly to the cortical surface. The arrow indicates a region where the cytoplasmic membranes of adjacent dendrites (D) are in close apposition. Magnification: x14 000.

 
The structure of the neuropil of Cx32-deficient mice resembled that of wild-type mice. The apical dendrites of pyramidal cells were arranged in vertical bundles. These bundles were most prominent in lamina IV, where they were formed by thick apical dendrites of large lamina V pyramidal cells (Fig. 2a,bGo). The bundles were also manifest in lamina II/III, where numerous small and medium sized pyramidal cells gave rise to smaller apical dendrites (Fig. 2a,bGo). Especially in sections obtained by tangential cuts through lamina IV (Fig. 2c,dGo) and lamina II/III (not shown), it became evident that the distribution pattern of dendritic bundles observed in Cx32-knockout animals did not differ from that of wild-type mice. Also at the ultrastructural level, no differences in the arrangement of dendritic bundles were encountered when comparing wild-type and Cx32- deficient mice (Fig. 2eGo). The apical dendrites forming one bundle were often separated from each other by fibrous neuropil consisting of thin unmyelinated axons, small dendritic branches, dendritic spines and irregularly shaped glial processes. Occasionally, apical dendrites were observed in direct apposition to each other (Fig. 2eGo). However, gap junctions were not detected in these regions of the neocortex.

In both experimental groups, the highest density of myelinated fibers was found in lamina VI (Fig. 3a,bGo). The distribution pattern of vertically and horizontally oriented fibers present in Cx32-null mutant mice (Fig. 3bGo) was identical to that of wild-type mice (Fig. 3aGo). Although the myelin sheaths of the axons of Cx32-deficient mice appeared to be somewhat blurred and less demarcated from their enviroment, light microscopical inspection revealed no obvious difference between the myelinated axons of wild-type mice and Cx32-null mutant mice (Fig. 3c,dGo).

We therefore investigated the structure of neocortical myelinated axons of both Cx32-knockout and wild-type mice by qualitative electron microscopy of myelin sheaths in all neo- cortical layers and in the white matter underlying the neocortex. Morphometrical analysis was restricted to myelinated axons located in the lower part of lamina VI, where the maximum density of myelinated fibers was found. Qualitatively, the compact structure of the axonal myelin sheaths of Cx32- deficient mice seemed to be similar to that of wild-type mice. However, differences were observed with respect to the periodicity of myelin: the distance between the major dense lines representing myelin lamellae was larger in Cx32-deficient mice as compared to wild-type animals (insets in Fig. 3e,fGo). In addition, in Cx32-null mutant animals, the lateral loops of the myelin lamellae in the paranodal regions appeared somewhat elongated and more irregular in shape and size than those in wild-type animals (Fig. 3e,fGo). Occasionally, asymmetrically protruding parts of the myelin sheath involved in the formation of the paranodal region were observed (Fig. 3fGo). These protrusions were filled with cytoplasm of the widened inner loop of the oligodendrocyte lamella. This electron microscopical appearance of the axons resembled that of the optic nerve during development of the nodes of Ranvier (Hildebrand and Waxman, 1984Go), suggesting an incomplete maturation of myelin sheaths in the neocortex of adult Cx32-deficient mice. Such irregular paranodal regions were never observed in wild-type animals.

The morphometrical analysis revealed a significant decrease (by ~19%, Table 1Go) in the volume fraction of myelin in the neocortical neuropil of Cx32-deficient mice. This decrease was associated with a significantly reduced thickness (by ~13%, Table 1Go) of the myelin sheath around a single axon. The average diameter of the axons (without myelin sheath) in the neocortex of Cx32-deficient mice was similar to that in wild-type animals. The density of myelinated axons appeared to be somewhat lower in the neocortex of Cx32-deficient mice as compared to wild-type animals. However, this difference was statistically not significant. The morphometrical parameters have been summarized in Table 1Go.

Electrophysiological Studies

Intracellular recordings were obtained from 30 neurons of Cx32- deficient mice (13 animals) and from 24 neurons of wild-type mice (13 animals). All neurons were located in neocortical layers II/III and could be classified as so-called regular spiking cells (McCormick et al., 1985Go). The tracer neurobiotin was injected intracellularly into 15 neurons and all of them were found to be pyramidal cells. Thus, in line with analyses performed previously in our laboratory (Rörig et al., 1996Go), it is legitimate to assume that the neuron population investigated in the present study consisted predominantly of pyramidal cells of neocortical layers II/III.

Subthreshold Membrane Properties, Action Potentials and Discharge Behavior

The electrophysiological parameters determined in neocortical neurons of wild-type mice and Cx32-deficient mice have been summarized in Table 2Go.


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Table 2 Comparison of electrophysiological parameters determined in neocortical neurons of wild-type and Cx32-deficient mice
 
There was no significant difference between the resting membrane potentials measured in control and in Cx32-knockout mice respectively (Table 2Go). The neuronal input resistances were calculated from the averages of steady-state voltage responses (n = 10) evoked by intracellular injections of hyperpolarizing current pulses (0.14 nA, 300 ms). In neurons of Cx32-deficient animals, the input resistance was found to be significantly increased by 27.4% as compared to that of neurons of wild-type mice (Table 2Go). The resting membrane potentials of the individual cells did not correlate with their input resistances, neither in neurons of Cx32-knockout mice (r2 = 0.11, n = 30) nor in those of wild-type animals (r2 = 0.00, n = 24). The ‘somatic’ membrane time constant, {tau}0, and the first equalizing time constant, {tau}1, were derived from voltage responses induced by hyperpolarizing current pulses. A curve defined by two exponential terms was fitted to the first 50 ms of the voltage transients (Rörig et al., 1996Go). The mean membrane time constant {tau}0 was similar in neurons of both experimental groups (Table 2Go). However, the first equalizing time constant {tau}1 was found to be significantly decreased (by ~20%) in neurons of Cx32-knockout mice (Table 2Go). The values of {tau}0 and {tau}1 of the individual neurons did not correlate with resting membrane potentials or input resistances, neither in wild-type animals nor in Cx32-deficient mice. Similarly, there was no correlation between {tau}0 and {tau}1.

The current–voltage (IV) curves of the neurons were determined by intracellular injection of depolarizing and hyperpolarizing current pulses (300 ms) of different amplitudes and measurement of the corresponding voltage deviations at the end of the pulses (steady-state IV curves). Figure 4a,bGo shows representative examples recorded from one neuron of each experimental group. ‘Representative’ means that resting membrane potentials and input resistances of the selected neurons were within a range of mean value ± 1 SD of the corresponding experimental group. In all neurons investigated (Cx32+/+: n = 20, Cx32–/–: n = 23), the IV curves displayed inward rectification in both de- and hyperpolarizing directions. However, in neurons of Cx32-deficient mice the slopes of the IV curves were found to be steeper than that of IV curves measured in neurons of wild-type mice (Fig. 4cGo). To quantify this difference, the slopes of the IV curves were determined at points corresponding to an injected current of –0.2 and +0.2 nA respectively. A rectification ratio was calculated by dividing the slope resistance obtained at +0.2 nA by the slope resistance determined at –0.2 nA. This analysis was performed with IV curves obtained from nine neurons recorded in slices of wild-type mice (nine different animals) and with IV curves from 11 neurons of Cx32-null mutant mice (11 different animals). There was no statistically significant difference between the mean resting membrane potentials of these two groups of neurons. At the selected points, the slopes of the IV curves were significantly larger in neurons of Cx32-deficient mice as compared to those of wild-type mice (Table 2Go). However, there was no significant difference between the rectification ratios (Table 2Go). This indicates that, within the potential range investigated, neocortical neurons of Cx32-deficient mice displayed at an evenly increased input resistance without changes in their rectifying properties.



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Figure 4. Current–voltage (IV) relationships and action potential discharge of neocortical neurons recorded from slices of wild-type mice and of Cx32-deficient mice. (a–c) The IV curves (a, Cx32+/+; b, Cx32–/–) were determined by injecting de- and hyperpolarizing current pulses (lower traces in a and b; for details see text). The subsequently recorded voltage responses (upper traces in a and b) are shown superimposed. The examples depicted in the figure were selected to be representative for each experimental group (see text). (c) Plot of the current-induced voltage changes ({Delta}V) as a function of the injected current amplitude ({Delta}I). (d,e) Responses of two neurons (d: Cx32+/+; e: Cx32–/–, upper traces) to depolarizing current pulses with an amplitude of 0.48 nA (300 ms duration, lower traces). (f,g) The diagrams show the discharge frequency as a function of the number of the interspike interval at different amplitudes of the injected current pulse. Examples in this figure are representative applying to the criteria given in the text (f: Cx32+/+, resting membrane potential: –85 mV, input resistance: 43.8 M{Omega}; g: Cx32–/–: resting membrane potential: –83 mV, input resistance: 51.5 M{Omega}).

 
Corresponding to the higher input resistances, the threshold current necessary to evoke a single action potential by intracellular injection of a 30 ms depolarizing current pulse was significantly smaller in neurons of Cx32-deficient mice (Table 2Go). Amplitude (96–107 mV) as well as duration (0.88–1.36 ms) of action potentials were similar in neurons of both experimental groups. Also, the depolarizing voltage changes necessary to reach spike threshold in neurons of Cx32-deficient mice did not differ from that determined in neurons of wild-type animals (26–32 mV).

The discharge behavior was investigated by injecting depolarizing current pulses of increasing amplitudes (Fig. 4dgGo). All neurons responded to injection of suprathreshold current pulses with repetitive action potential discharge associated with frequency adaptation (Fig. 4f,gGo). However, comparison of neurons matched to the mean resting membrane potential and mean input resistance of each experimental group revealed that the discharge frequency induced by a given current strength was higher in neurons of Cx32-deficient mice (Fig. 4f,gGo). In order to quantify this difference, we determined the mean frequency of the last two interspike intervals of a spike train and plotted it as a function of the injected current (f–I plots). The slopes of the resulting lines were determined by linear regression. This analysis was performed with recordings obtained from 11 neurons (from 11 different mice) of each experimental group with mean resting membrane potentials which were not significantly different. The mean slope of the f–I plots derived from current-induced steady-state discharge was found to be significantly increased (by 24%) in neurons of Cx32-deficient mice compared to that obtained from neurons of wild-type mice (Table 2Go).

Stimulus-evoked Postsynaptic Potentials

The postsynaptic stimulus-response pattern was analysed in 16 neurons of wild-type mice (seven different animals) and in 20 neurons of Cx32-null mutant mice (nine different animals). The stimulation electrode was positioned into the white matter underlying the neocortex. Recordings were taken from neurons lying approximately on a vertical line between stimulation electrode and pial surface of the slice. In order to determine the stimulus–response behavior of a neuron, the stimulus strength was gradually increased in increments of 25 µA starting from the threshold intensity necessary to evoke a measurable and stable postsynaptic potential. Neurons of control mice responded to stimulation with increasing intensity in a similar way as neurons of the rat neocortex (Sutor and Hablitz, 1989aGo,bGo): at low intensities, short-latency EPSPs (early EPSPs, e-EPSP) with small amplitudes and short durations were evoked (Fig. 5aGo, 150 µA and Fig. 5cGo, 125 µA). An increase in stimulus strength led to an increase in the amplitude of the e-EPSP (Fig. 5aGo, 250 µA) and, in 9/16 neurons, to the elicitation of a so-called late EPSP (l-EPSP, Fig. 5cGo, 150 and 200 µA, arrows). Further enhancement of the stimulus intensity induced suprathreshold EPSPs and depolarizing fast IPSPs (f-IPSP, Fig. 5aGo, 600 µA and Fig. 5cGo, 400 µA). Following experimental depolarization of the membrane potential to various levels, the amplitudes of the f-IPSPs decreased and additional hyperpolarizing late IPSPs (l-IPSPs) became evident (Fig. 5bGo). The mean reversal potential of the f-IPSPs was –66.4 ± 6.5 mV (n = 5) and that of the l-IPSP was –88.0 ± 4.5 mV (n = 5) (Fig. 5bGo). This stimulus–response pattern was observed in all neurons investigated in slices of wild-type mice. The properties of the l-EPSPs detected in ~50% of the neurons of wild-type mice were identical to those of l-EPSPs described in rat neocortical neurons (Sutor and Hablitz, 1989aGo,bGo).



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Figure 5. Evoked postsynaptic potentials recorded from neocortical neurons of wild-type (a–c, Cx32+/+) and Cx32-deficient mice (d, Cx32–/–). (a) Low-intensity stimuli elicited subthreshold depolarizing postsynaptic potentials with graded amplitude changes (150 and 250 µA). At higher intensities (600 µA), the stimuli evoked suprathreshold EPSPs followed by slowly decaying depolarizing IPSPs. (b) Determination of the reversal potential revealed the presence of a fast IPSP (reversal potential: –72 mV) and a late IPSP (reversal potential: –93 mV). Recordings were taken subsequently at the membrane potentials indicated and are displayed superimposed. (c) In ~50% of the neurons of wild-type animals, stimulation at medium intensities (150 and 200 µA) evoked a so-called late EPSP (l-EPSP, arrows). Note the almost complete disappearance of this l-EPSP at stimulus strengths sufficient to evoke a depolarizing IPSP (400 µA). (d) Eleven out of 20 neurons recorded from slices obtained from Cx32-deficient mice responded to stimulation with an all-or-nothing depolarizing potential change (in this case associated with high frequency action potential discharge). Increasing the stimulus intensity led to a decrease in the latency of the bursts without changing their magnitude. All traces in (a–d) represent three subsequently recorded traces superimposed. In this and all other figures, the time point of stimulation is indicated by an arrow.

 
Eleven of the 20 neurons recorded from slices obtained from Cx32-deficient mice displayed a synaptic stimulus-response pattern similar to that shown in Figure 5dGo: at threshold intensity (Fig. 5dGo, 125 µA), the neurons generated large depolarizing potential changes (at resting membrane potential: 40–50 mV in amplitude and 150–200 ms in duration) associated with either high-frequency action potential discharge (Fig. 5dGo) or with the generation of a few single spikes (see Figs 6 and 7GoGo). The latencies of these responses ranged between 20 and 50 ms and an increase in stimulus intensity led to a decrease in latency without changes in the magnitude of these potentials (Fig. 5dGo, 150 and 200 µA). Thus, these evoked depolarizing potential changes displayed an all-or-nothing behavior.



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Figure 6. (a,b) Long-latency afterdischarges in neurons of Cx32-deficient mice. At a frequency of 0.05 Hz and a stimulus strength of 125 mA (a, upper trace), this neuron responded to stimulation with a two-component subthreshold EPSP. An increase in the intensity to 150 mA (a, lower trace) induced early bursts and, in one out of three trials, a late afterdischarge. Following a decrease in stimulus frequency to ~0.017 Hz, stimulation with 125 mA (b, upper trace) induced two initial bursts and one afterdischarge in five trials. Upon an increase in stimulation intensity (150 mA), every stimulus reliably evoked both an early burst and a late afterdischarge (b, lower trace). The single trials are presented superimposed. (c) Nine out of 20 neurons recorded from Cx32-deficient mice displayed a stimulus–response behavior very similar to that observed in wild-type animals (see Fig. 5aGo). In each trace, three subsequently taken recordings are shown superimposed. (d) Determination of the IPSP reversal potentials in a neuron from a Cx32-null mutant mouse (see Fig. 5bGo). Reversal potential of the fast IPSP: –74 mV; reversal potential of the late IPSP: –91 mV.

 


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Figure 7. Action of the NMDA receptor antagonist D-2-APV (a, 20 mM), the AMPA receptor antagonist CNQX (b, 5 mM), and the GABAA receptor antagonist bicuculline (c, 10 mM) on the evoked late depolarizing potentials observed in neurons of Cx32-deficient mice. Both glutamatergic antagonists reversibly inhibited these responses (a,b). Application of bicuculline led to the induction of a paroxysmal depolarization which could be blocked by D-2-APV and CNQX (c). In (a,b), each trace represents three single recordings superimposed. In (c), single records are shown.

 
Two of the 11 neurons with altered postsynaptic potentials generated late responses with latencies of ~1 s (Fig. 6aGo). These late responses were sensitive to stimulus frequency. At a stimulation intensity of 125 µA and a frequency of 0.05 Hz, the neuron depicted in Figure 6aGo (upper traces) responded with small subthreshold EPSPs consisting of an early and a late component. An increase in the stimulus strength to 150 µA induced a large initial burst response and, in one out of three trials, a delayed afterdischarge (Fig. 6aGo, lower traces). Upon reduction of the stimulus frequency to one stimulus per min (~0.017 Hz), early and late burst discharges were occasionally elicited at a stimulus strength of 125 µA (Fig. 6bGo, upper traces, five trials), but were reliably evoked by every stimulus at an intensity of 150 µA (Fig. 6bGo, lower traces, five trials).

Nine of the 20 neurons investigated in slices obtained from Cx32-deficient mice revealed a stimulus–response pattern similar to that of control mice (Fig. 6cGo). In five of these cells, the reversal potentials of the IPSPs were determined (Fig. 6dGo, f-IPSP: –69.6 ± 6.8 mV; l-IPSP: –89.2 ± 1.6 mV). The reversal potentials were statistically not different from those measured in neurons of control mice.

The described alterations in synaptic response behavior were observed in slices prepared from the neocortices of seven (out of nine, see above) different Cx32-deficient mice. It is important to note that neurons with altered stimulus–response patterns could be found in the same slice of a Cx32-null mutant mouse in which neurons with normal stimulus–response behavior were observed. Furthermore, in slices of Cx32-deficient mice, the mean input resistance of neurons with altered postsynaptic potentials was slightly larger (64.00 ± 19.21 M{Omega}, n = 11, versus 55.89 ± 16.90 M{Omega}, n = 9) and their mean first equalizing time constant {tau}1 was somewhat smaller (1.36 ± 0.51 ms, n = 11, versus 1.41 ± 0.53 ms, n = 9) as compared to neurons of Cx32-deficient mice with normal synaptic responses. However, these differences were statistically not significant. Thus, the altered pattern of post- synaptic potentials observed in 11 out of 20 neurons of Cx32- deficient mice cannot be attributed to a different electrotonic structure of these cells.

The evoked late depolarizing potentials observed in neurons of Cx32-deficient were reversibly blocked by application of NMDA as well as AMPA receptor antagonists (D-2-APV, 10–20 µM, n = 5, Fig. 7aGo; CNQX, 5–10 µM, n = 4, Fig. 7bGo). These data indicate that the late depolarizing potential changes observed in neurons of Cx32-deficient mice were mediated by glutamatergic synapses and that activation of both NMDA receptors and AMPA receptors was essential for their generation.

In all neurons of Cx32-deficient mice which were recorded using microelectrodes filled with potassium acetate and which displayed an altered synaptic response pattern (n = 6), GABAergic inhibition was virtually absent. Even at high intensity stimulation, it was not possible to elicit detectable IPSP components. However, as shown in Figure 7cGo, the late depolarizing potential changes were generated in the presence of substantial GABAA receptor-mediated inhibition within the neuronal network. Application of the GABAA receptor antagonist bicuculline led to a prolongation of these potentials and to the generation of repetitive afterdischarges (Fig. 7cGo, n = 3). These paroxysmal depolarizations could be completely blocked by D-2-APV and CNQX (Fig. 7cGo, n = 3).

The amplitudes of the evoked late depolarizing potentials increased upon hyperpolarization and decreased following depolarization of the membrane potential (Fig. 8Go). In addition, they were sensitive to stimulus frequency. At a frequency of 0.05 Hz, each of ten subsequent stimuli evoked a fully developed response (Fig. 9aGo). However, only two out of ten succeeding stimuli were effective at eliciting a burst discharge following an increase in stimulus frequency to 0.5 Hz (Fig. 9bGo, n = 4).



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Figure 8. Voltage dependence of the late burst responses evoked in neurons of Cx32-deficient mice. Intracellular recordings from a neuron with a resting membrane potential of –86 mV. At different membrane potentials adjusted by intracellular current injection, a late burst response was induced by stimulation at an intensity of 175 mA. The amplitude underlying the burst increased with hyperpolarization and decreased with depolarization. In each trace three single recordings were superimposed. Note the stimulus-induced irregular discharge of small depolarizing potentials. Recordings were performed with a microelectrode filled with 3 M KCl. In this figure, action potentials have been truncated.

 


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Figure 9. The late burst discharges evoked in neurons of Cx32-deficient mice could not follow stimulation frequencies >0.5 Hz. At a stimulation frequency of 0.05 Hz, 10 out of 10 subsequent stimuli successfully elicited burst discharges (a). At a frequency of 0.5 Hz, only 2 out of 10 stimuli were able to evoke a burst discharge (b). Similar to the initial burst responses, the small depolarizing potentials disappeared upon an increase in stimulus frequency (b). Recordings were performed using a microelectrode filled with 3 M KCl. Resting membrane potential: –86 mV.

 
When recorded with electrodes filled with 3 M KCl, all neurons (Cx32+/+: n = 3; Cx32–/–: n = 5), independent of the genotype, spontaneously generated small (3–12 mV) depolarizing synaptic potentials (DSPs) at an average rate of 1–2 events/s. In four out of five neurons investigated in slices from Cx32-null mutant mice, electrical stimulation increased the rate of occurrence of these DSPs by a factor of 5–15 within a time window of 1–2 s following the burst response (Fig. 10aGo). This stimulus-correlated phenomenon was not detected in neurons of wild-type mice. In the presence of an evoked burst (i.e. at medium to high stimulus intensities), the rate of the DSPs increased with stimulus strength (Fig. 10aGo, 175 and 400 µA). The application of bicuculline led to the disappearance of this spontaneous depolarizing potentials (Fig. 10bGo) identifying these DSPs as spontaneous IPSPs mediated via the GABAA receptor. Their depolarizing time course was due to the increased intracellular chloride concentration induced by the diffusion of chloride out of the recording electrodes. The generation of these stimulus-correlated, but asynchronous IPSPs was dependent on the presence of a stimulus-evoked burst response. When the burst response was suppressed by D-2-APV (Fig. 10cGo) or by increasing stimulus frequency (see Fig. 9Go), the DSPs disappeared. Thus, these DSPs can be characterized as small, irregularly occurring GABAA receptor mediated IPSPs reflecting prolonged stimulus-evoked synaptic release of GABA.



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Figure 10. Properties of stimulus-correlated asynchronous depolarizing synaptic potentials (DSPs) observed in neurons of Cx32-null mutant mice during intracellular recordings by means of microelectrodes filled with 3 M KCl. (a) The DSPs occurred stimulus-correlated within a time window of 1–2 s post-stimulus. Their frequency depended on the stimulus intensity. (b) The DSPs were blocked by the GABAA receptor antagonist bicuculline (10 mM). (c) The DSPs disappeared following inhibition of the late burst response by the NMDA receptor antagonist D-2-APV. The effects of D-2-APV were reversible upon wash out of the compound. Recordings shown in (a,c) were obtained from the same neuron. In (b,c), three superimposed single recordings are depicted in each trace. Action potentials have been truncated.

 
Spontaneous Synaptic Activity

All neurons of Cx32-deficient mice with altered stimulus– response behavior generated spontaneous burst discharges, either as single bursts (Fig. 11aGo) or as burst doublets (Fig. 11bGo). These bursts had a duration of 500–800 ms, occurred at a frequency of 2–8/min and were never observed in neurons of wild-type mice. They were also not observed in neurons of Cx32-deficient mice with unaltered stimulus–response pattern.



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Figure 11. Spontaneous burst discharges recorded from two neurons of different Cx32-deficient mice. The recordings were taken continuously over a period of 1 min. (a) This neuron generated single bursts at a frequency of 5–8/min. (b) In this neuron, burst doublets were observed at a frequency of 2–4/min.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
In the rodent CNS, Cx32 has been found predominantly in oligodendrocytes (Dermietzel et al., 1989Go; Scherer et al., 1995Go; Nadarajah et al., 1996Go). These glial cells are responsible for axon myelination (Parnavelas et al., 1983Go). During postnatal development of the rat neocortex, detectable Cx32 protein expression starts around postnatal day 5–7 (Dermietzel et al., 1989Go; Nadarajah et al., 1997Go). The Cx32 levels steadily increase with ongoing development until they achieve a maximum around postnatal day 20 (Nadarajah et al., 1997Go). This expression time course matches almost exactly that of oligodendrocyte maturation (Parnavelas et al., 1983Go) and myelination commences approximately at postnatal day 10 (Parnavelas et al., 1983Go). The very close temporal correlation between Cx32-expression, oligodendrocyte maturation and commencement of myelination suggests an involvement of this gap junction protein in the generation of myelin sheaths around neocortical axons (Parnavelas et al., 1983Go). Thus, we primarily expected impaired conduction properties of neocortical axons in Cx32-deficient mice. We have tried to determine the single axon conduction velocity in neocortical slices by means of extracellular recordings. However, these experiments were not successful. In slice preparations, it is almost impossible to unambiguously assign stimulus-evoked action potentials recorded at two different sites to the same single fiber.

We observed no differences in the number of axons per unit area when comparing neocortices of Cx32-deficient mice with those of wild-type animals. Therefore, we conclude that the reduction (by 19%) in volume fraction of myelin in the neocortex of Cx32-deficient mice was mainly due to thinner myelin sheaths around single axons. In contrast to studies performed in the PNS, (Anzini et al., 1997Go; Scherer et al., 1998Go), we did not detect consistent changes in the compact structure of myelin as well as in the stucture of the nodes of Ranvier. The distance between two major dense lines of the myelin sheath was somewhat larger in Cx32-deficient mice and in some of the nodes of Ranvier encountered, the end loops appeared in an abnormal shape. Thus, our results indicate weak disturbances in myelination leading to dysmyelination (Bostock, 1993Go) of neocortical axons in Cx32-deficient mice. Dysmyelination may result in a decrease in fiber conduction velocity. Based on an axon model, W.A.H. Rushton defined a critical ratio g (ratio of the axon diameter to the overall fiber diameter) which is required for optimal conduction velocity (Rushton, 1951Go). In peripheral nerves, the optimal value of g is 0.6. Waxman and Bennett reported values between 0.5 and 0.7 for myelinated CNS fibers (Waxman and Bennett, 1972Go). Analysis of our morphometrical data revealed that in both wild-type mice (0.62) and Cx32-deficent mice (0.64), the ratio g was found to be in the optimal range. Therefore, we assume that the small reduction (by 13%) in the thickness of the myelin sheath does not severely affect conduction velocity of myelinated fibers in the neocortex of Cx32-deficient mice.

One explanation for the minor alterations in myelination of neocortical axons might be that, although expressed in oligo- dendrocytes, Cx32 is not primarily involved in the generation of axonal myelin sheaths. However, taking into consideration that myelination of peripheral nerves is severely disturbed in Cx32-deficient mice (Anzini et al., 1997Go; Scherer et al., 1998Go), this explanation seems to be unlikely. Another possibility to explain our observations is that the function of Cx32 is compensated for by another connexin expressed in oligodendrocytes of wild-type and Cx32-deficient mice. There is evidence for the co-expression of Cx32 and Cx45 in these glial cells (Kunzelmann et al., 1997Go). Thus, Cx45 is a candidate which might at least partially neutralize the lack of Cx32 in oligodendrocytes of Cx32-null mutant mice.

In another study performed on Cx32-deficient mice, no changes in myelination of the optic nerve have been described (Scherer et al., 1998Go). Similarly to Scherer et al., we did not observe consistent alterations in the qualitative appearance of the compact myelin structure as well as in the structure of the nodes of Ranvier. Our conclusion that neocortical axons of Cx32-null mutant are dysmyelinated results predominantly from a morphometrical analysis (volume fraction of myelin, etc.). Such morphometric data are not available for the optic nerve.

In addition to oligodendrocytes, experimental evidence exists for the expression of Cx32 in neocortical neurons (Dermietzel et al., 1989Go; Nadarajah et al., 1997Go). It is not clear which types of neocortical neurons generate gap junctions formed by Cx32. However, in the mature neocortex, the incidence of gap junction-mediated coupling among pyramidal cells is very low (Connors et al., 1983Go; Peinado et al., 1993Go; Rörig and Sutor, 1996Go). Thus, it seems to be very unlikely that a lack of Cx32 in mature pyramidal cells is the imminent cause of the alterations in intrinsic properties (including cell geometry) and synaptic function which we have observed in neurons of Cx32-deficient mice. During the first two weeks of postnatal development of the neocortex, however, the incidence of gap junction-mediated coupling among pyramidal neurons is high and, within the period from postnatal day 5–15, neuronal coupling and Cx32- expression coincide. Thus, a transient expression of Cx32 in pyramidal cells during postnatal development of the neocortex cannot be excluded. Neurons recorded from slices of Cx32- deficient mice displayed significant changes in electrotonic properties which resulted in an enhanced intrinsic excitability. These changes occurred independently of the neurons' synaptic response patterns. Our observations suggest the existence of as yet unknown mechanisms which depend on the presence of neuronal Cx32 and which influence the maturation of intrinsic properties of neocortical pyramidal cells during the early period of postnatal development.

In the developing neocortex, gap junctions have been shown to be involved in the generation of so-called neuronal domains (Yuste et al., 1992Go, 1995Go). It has been suggested that these domains represent a blueprint for the adult functional architecture of the neocortex (Yuste et al., 1992Go). Cx32-deficient mice did not display alterations in the basic pattern of lamination and in the structure of the vertical compartmentation [i.e. dendritic bundling (Schmolke, 1989Go, 1996Go)]. This observation is in accordance with the fact that Cx32 is virtually not present during the developmental period of cell fate decision, neurogenesis and migration (Miller, 1988Go; Peters, 1993Go; Nadarajah et al., 1997Go). Thus, Cx32 is probably not involved in the formation of neuronal domains during postnatal development of the neocortex.

The most unexpected finding in our study was a strong potentiation of glutamatergic synaptic transmission in ~50% of the neurons investigated in slices of Cx32-deficient mice. Orthodromic stimulation evoked late depolarizing potentials mediated by the NMDA- and AMPA-subtype of the glutamate receptor. The magnitude of these potentials varied considerably in different cells. In some neurons, they resembled paroxysmal depolarizations induced in neocortical neurons by application of GABAA receptor antagonists (Gutnick et al., 1982Go; Lee and Hablitz, 1991Go). In other neurons, they appeared to be similar to the so-called l-EPSP which has been described in layer II/III neurons of the rat neocortex (Sutor and Hablitz, 1989aGo,bGo; Hwa and Avoli, 1992Go) and in neocortical neurons of wild-type mice (see Fig. 5cGo). However, the late depolarizing potentials observed in neurons of Cx32-deficient mice differed from l-EPSPs in three aspects: (i) their amplitudes were considerably larger and their durations as well as their latencies (at threshold intensities) were longer lasting; (ii) they displayed an all-or-nothing behavior; and (iii) they did not disappear at high stimulation intensities. In neocortical neurons of rat and wild-type mice, the l-EPSPs disappeared at high stimulus strengths due to the elicitation of efficient GABAergic IPSPs. Such IPSPs were virtually absent in neurons of Cx32-null mutant mice. Thus, we conclude that these late depolarizing potentials represent strongly expressed EPSPs generated in the absence of efficient neuronal inhibition. Similar large EPSPs have been described in neocortical neurons in the presence of low concentrations of GABAA receptor antagonists (Chagnac-Amitai and Connors, 1989Go; Lee and Hablitz, 1991Go).

Recordings using microelectrodes filled with 3 M KCl revealed that neurons of Cx32-deficient mice with altered synaptic response patterns still received GABAergic input. It consisted of a stimulus-correlated asynchronous discharge of small IPSPs occurring within a time window of 1–2 s post-stimulus. However, these small asynchronous IPSPs were insufficient to control synaptically evoked excitation. This observation indicates a defective GABAergic system in the neocortex of Cx32-deficient mice. Some of our results obtained from neurons of Cx32-deficient mice, however, exclude a defect which affects the GABAergic system in general: (i) altered synaptic response patterns were observed in only 50% of the neurons investigated; (ii) neurons with normal and abnormal synaptic potentials could be found in the same slice; and (iii) application of bicuculline produced paroxysmal depolarizations in all neurons tested, indicating the presence of substantial GABAA receptor-mediated inhibition within the neocortical network.

Due to the lack of experimental data on the cell-specific expression of Cx32 in neocortical pyramidal cells and/or interneurons, we cannot provide, at least at the moment, a comprehensive explanation for our observations. However, it has recently been reported that inhibitory interneurons of the rat neocortex are organized as local networks generating synchronous activity (Galarreta and Hestrin, 1999Go; Gibson et al., 1999Go). The synchronization of these networks is mediated via gap junctions connecting the interneurons. If Cx32 contributes to intercellular connections between neocortical inhibitory interneurons, it is reasonable to assume that a lack of Cx32 results in a desynchronization of the network activity. Desyn- chronization of an inhibitory network would be a possible explanation for the stimulus-correlated asynchronous discharge of short-lasting GABAA receptor-mediated IPSPs which we observed in neocortical neurons of Cx32-deficient mice. Furthermore, Gibson et al. described two functionally distinct inhibitory networks within the neocortex (Gibson et al., 1999Go). Thus, one might speculate that Cx32 is involved in the synchronization of only one of these networks. As a consequence, a lack of Cx32 would not affect neocortical GABAergic inhibition in toto which might explain our observation that only ~50% of the neurons investigated displayed evoked late depolarizing potentials.

In the neocortex, a lack of Cx32 is correlated with dysmyelination of axons, alterations in intrinsic neuronal properties and defects in GABAergic synaptic transmission. These findings indicate that gap junctions formed by Cx32 are important factors in the regulation of the function of neocortical neurons, networks and glia cells. We do not want to exclude disturbances of other cellular and network functions associated with a ‘knockout’ of Cx32 (e.g. changes in metabolism or alterations in developmental mechanisms). However, they seem to be not as obvious as the defects which we have observed. In order to understand the significance of Cx32, it will be necessary to investigate the cell-type specific expression of Cx32 and to identify neuronal and glial networks within the neocortex the functions of which depend on gap junctions formed by Cx32.


    Notes
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Notes
 References
 
We thank Dr Thomas Ott (Institute of Genetics, Bonn) for help with genotyping the mice, G. Everloh, A. Ihmer, G. Kruse (Institute of Anatomy, Bonn) for technical assistance and Dr M. Knapp (Institute of Medical Statistics, Bonn) for statistical analysis of the morphological data. This work was supported by grants of the Deutsche Forschungsgemeinschaft (Su 104/3–1, Wi 270/22–1, SFB 400/B3), by the Friedrich-Baur-Stiftung (to B.S.) and by the Fonds der Chemischen Industrie (to K.W.).

Address correspondence to Bernd Sutor, Institute of Physiology, University of Munich, Pettenkoferstrasse 12, D-80336 München, Germany. Email: bernd.sutor{at}lrz.uni-muenchen.de.


    Footnotes
 
2 Present address: Rheinische Kliniken Bonn, Kaiser-Karl-Ring 20, D-53111 Bonn, Germany Back


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