AG Entwicklungsneurobiologie, Fakultät für Biologie, Ruhr-Universität, ND 7/31, D-44780 Bochum, Germany
Address correspondence to P. Wahle, AG Entwicklungsneurobiologie, Fakultät für Biologie, Ruhr-Universität, ND 7/31, D-44780 Bochum, Germany. Email: wahle{at}neurobiologie.ruhr-uni-bochum.de.
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Abstract |
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Introduction |
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Further, BDNF production depends on activity. BDNF mRNA transcription requires calcium influx through voltage-sensitive channels evoked by depolarizing stimuli (Shieh et al., 1998; Tao et al., 1998
). BDNF increases after activation of glutamate receptors (Zafra et al., 1990
, 1991
; Wetmore et al., 1994
), and during seizures or spreading depression which, for instance, do not increase NT-3 expression (Kokaia et al., 1993
; Nawa et al., 1995
). Accordingly, sensory cortical BDNF expression depends on sensory stimulation [light in visual cortex (Castrén et al., 1992
; Bozzi et al., 1995
); whisker stimulation in barrel cortex (Rocamora et al., 1996
)]. Moreover, BDNF is sorted into the regulated secretory pathway and released in an activity-dependent way (Lindholm et al., 1994
; Androutsellis-Theotokis et al., 1996
; Goodman et al., 1996
; Haubensak et al., 1998
). Once released, neurotrophins may stimulate their own release (Canossa et al., 1997
) and the release of other neurotransmitters (Sala et al., 1998
) and thus influence neuronal activity (Berninger and Poo, 1996
). With regard to BDNF expression previous studies have mainly focused on mechanisms of BDNF upregulation due to experimentally evoked increases in activity in immature dissociated neuron cultures or by pathophysiological activity in adult cortex. Less is known about the regulation of BDNF by spontaneous activity intrinsic to a cortical neuron network, and it is, for instance, unknown how much activity is required for the developmental upregulation and adult steady-state expression levels of BDNF.
Here we focused on the role of SBA in regulating cortical neurotrophin expression using organotypic cultures (OTCs) (Gähwiler et al., 1997) of rat visual cortex. OTCs conserve the laminar neighborhood and organotypic ratios of neuronal types. This allows for proper differentiation of morphology, intrinsic circuitry and long survival periods (Bolz et al., 1990
, Götz and Bolz 1994
; Obst and Wahle, 1995
, 1997
). OTCs develop an appreciable level of synaptically generated spontaneous activity in the absence of afferents and sensory input (Caeser et al., 1989
; Plenz and Aertsen, 1996
; Klostermann and Wahle, 1999
), and activity levels can be easily manipulated. The present study was designed to correlate the developmentally occurring and experimentally induced changes of activity with changes in expression of the neurotrophins BDNF, NT-3, NT-4/5 and their receptors. We analyzed the development and cell-type-specific patterns of SBA in OTCs, and the changes occurring during recovery from activity deprivation. For comparison, BDNF mRNA expression in developing visual cortex in vivo was analyzed. Specifically, we ask the following questions. (I) Is the intrinsically generated activity in cortex cultured without thalamic or sensory input sufficient for upregulation and longterm maintenance of neurotrophin and neurotrophin receptor expression? (ii) Is the expression of neurotrophins and their receptors altered by a chronic or transient activity blockade? We report here that OTCs develop a well-balanced state of excitation and inhibition. Activity deprivation appears to have little influence on structural maturation and on NT-4/5, NT-3, tyrosine kinase receptor C (trkC) and trkB mRNA receptor expression, but a dramatic influence on BDNF mRNA. The BDNF mRNA expression in immature and in differentiated cortical neurons remains tightly coupled to SBA.
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Materials and Methods |
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OTCs of rat visual cortex were prepared from LongEvans hooded rats at the day of birth essentially as described previously (Obst and Wahle, 1995, 1997
; Wirth et al., 1998
). OTCs were supplied with 750 ml of semi-artificial medium (50% BME, 25% HBSS and 25% horse serum, 1% glucose, 1 mM L-glutamine; all from Gibco). At 1 day in vitro (DIV), OTCs were incubated for 24 h with an anti-mitotic cocktail to prevent glial proliferation. To block SBA, medium containing a final concentration of 10 mM magnesium sulfate (J.T. Baker) was applied from 2 or 3 DIV onwards for the time periods indicated in the text. Medium was exchanged every 3 or 4 days. For recovery, cultures were returned to normal medium containing 2 mM magnesium for the indicated time periods. In some recovery experiments the cultures were exposed to 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX a non- NMDA antagonist; 20 µM, Tocris Cookson), D,L-2-amino-5-phosphovaleric acid (APV a NMDA receptor antogonist; 100 µM, Sigma) or nifedipine (a L-type Ca2+ channel blocker; 50 µM, Bayer) for the time periods indicated in the text.
Electrophysiology
Recordings were performed using conventional intracellular methods. Glass microelectrodes were pulled on a horizontal puller (Sutter Instruments) from borosilicate glass pipettes (o.d. 1.5 mm) and filled with 2 M potassium acetate and 1% Biocytin (Sigma). Electrode resistence ranged from 50 to 100 M. The coverslip with the culture was mounted into a glass-bottomed recording chamber and superfused (12 ml/min) with artificial cerebrospinal fluid (ACSF, pH 7: 124 mM NaCl, 5 mM KCl, 1.25 mM Na2H2PO4, 26 mM NaHCO3, 2 mM MgSO4, 2 mM CaCl2, 10 mM glucose) continuously bubbled with a mixture of 95% O2 and 5% CO2. Temperature was kept at 34°C. The recording chamber was mounted on an inverted microscope (Zeiss). Electrodes were positioned under visual control with a hydraulic drive (Narishige 3D Waterrobot), and neurons were impaled using a short decompensation of the electrode capacity. Intracellular potentials were recorded with an active bridge amplifier (Dagan). After stabilization of the membrane potential (~30 min), spontaneously occurring action potentials were recorded over a period of 10 min using a spike discriminator build in the A/D converter. The spontaneous activity was expressed as impulses per second (I/s). Intrinsic physiological parameters of recorded neurons (time constant, input resistance) were determined by injecting de- and hyperpolarizing current pulses of 150 ms duration (Master-8 stimulator, AMPI) in the range 0.12 nA. Spike amplitude and duration at half maximum were measured at spontaneous action potentials. Resting membrane potential was calculated by subtracting the resting intracellular voltage from the extracellular voltage measured when the electrode was withdrawn from the cell. Data were stored on a PC after digitization with an A/D converter (1401 CEDplus, Cambridge Electronic Design). Criteria for stable recordings were: cells had stable membrane potentials for 30 min up to 5 h, all were able to generate multiple action potentials on depolarizing current injection, and action potential amplitudes were >50 mV. Furthermore, all recorded cells showed spontaneously occurring postsynaptic potentials which were either excitatory (EPSPs) or inhibitory. In a majority of the neurons in OTC older than 20 DIV, the summation of EPSPs resulted in membrane potential deflection reaching a threshold, leading to action potentials. These action potentials, generated by synaptic input and without experimental stimulation, were called spontaneous.
Biocytin staining
After recording biocytin was injected with 100 ms depolarizing current pulses with 1 nA amplitude at a frequency of 1 Hz for 10 min plus 24 h for transport of biocytin. Cultures were fixed for 1 h in 4% para-formaldehyde in 0.1 M phosphate-buffered saline, pH 7.4 (PBS). Cultures were processed by the avidin-biotinylated horseradish peroxidase (ABC) method (Hsu et al., 1981). After three 10 min washes in PBS, followed by 0.5% Triton X-100 in PBS for 20 min, cultures were incubated in ABC solution (1:100, Vector Laboratories) at 4°C for at least 12 h. After washing in PBS and TrisHCl buffer (TB, pH 7.4), peroxidase activity was developed with 0.03% diaminobenzidine (Sigma) in TB and 0.005% hydrogen peroxide for 20 min under visual control, and stopped in TB. In some cultures, staining was intensified with 0.1% osmium tetroxide in PBS for 1 min. Cultures were dehydrated, cleared and embedded in Eukitt. The morphology of biocytin-stained neurons was reconstructed with a computer-based microscope system (Eutectic Electronics, Inc., Raleigh, NC, USA).
Generation of cDNA Libraries and PCR
Messenger RNA was isolated from visual cortex areas 17/18 of rats killed by an overdose of pentobarbitone (Nembutal, 60 mg/kg) at the ages given in Figure 5A, and from OTCs at several stages of development (given in DIV, indicated in Figs 5B and 6
). For each time point or experimental condition analyzed, five OTCs were harvested, pooled and mRNA was isolated using a DynaBead mRNA Direct Kit (Dynal). cDNA was synthetized with AMV reverse transcriptase (20 U/ml, Stratagene) at 42°C for 45 min. Semiquantitative PCR was performed in a total volume of 50 ml with GoldStar DNA Polymerase (0.5 U/ml; Eurogentec). Amplified regions (in base pairs) were as follows: BDNF I, 154393; BDNF II, 439823 (both specific for 3' exon V) and NT-3, 158634 (Maisonpierre et al., 1991
); NT-4/5, 309605 (Berkemeier et al., 1991
); trkB, 14221931 (specific for the kinase domain) (Middlemas et al., 1991
); trkC, 186739 (Valenzuela et al., 1993
). Glucose-6-phosphate dehydrogenase (G6PDH: 21122272) (Ho et al., 1988
) was chosen as a standard, because it is constitutively expressed throughout development and lacks induction by activity (Inokuchi et al., 1996
). The number of cycles was kept in the linear range determined for each product. To compare the developmental profile of BDNF mRNA expression in vivo and in OTCs, identical polymerase chain reaction (PCR) conditions and cycle numbers were run. The reactions were densitometrically analyzed with the Eagle Eye system (Stratagene), and normalization to G6PDH was used to determine the relative intensity of the bands. Normalized values from at least three PCR reactions were used to construct the graphs with standard deviation from mean.
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The PCR primers contained restriction sites for directional cloning of BDNF II, trkB and trkC. The products were cloned into pBS+/ (Strata-gene) and used to generate DIG-UTP-labeled riboprobes (Boehringer Mannheim). In situ hybridization was performed on free-floating OTCs at 55°C followed by stringent washes with sense hybridizations remaining negative, as detailed elsewhere (Obst and Wahle, 1995; Gorba and Wahle, 1999
). For every experimental condition, at least two hybridizations each with five OTCs from different batches were performed. OTCs were analyzed and photographed with a Zeiss Axiophot microscope on black and white film.
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Results |
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The physiological analysis is based on 153 neurons from >120 monocultures. The firing patterns of the neurons differentiate from intermediate spike patterns to class-specific patterns during the second week (Massengill et al., 1997). We therefore started to record from 12 DIV up to 168 DIV. All recorded neurons included here were classified as pyramidal cells. Passive and active membrane properties were summarized in Table 1
. In developing OTCs synaptically generated spontaneous activity was low at 12, 15 and 19 DIV (Fig. 1A
). Only 5/14 cells (38%) recorded and filled during this period displayed spontaneous action potentials at a mean rate of 0.03 I/s. All, however, generated trains of multiple action potentials when stimulated by current injections (insets in Fig. 1AD
). Significantly higher spontaneous activity levels with a mean rate of 0.20 I/s (highest rates were ~1.2 I/s) were recorded in OTCs older than 20 DIV (MannWhitney U-test, P < 0.05). Now 115/139 cells (83%) displayed action potential activity, and this did not change over age in vitro (Table 1
). Cells displayed single spikes or groups of very few spikes riding on EPSPs (Fig. 1BD
). Upon current injection, a majority of pyramidal cells from the second week onwards displayed a regular firing pattern with adapting spike trains (Fig. 1E1
). Only four neurons (Fig. 1E2
) were similar to intrinsic burst (IB) or doublet firing pyramidal neurons (McCormick et al., 1985
, 1993
; Connors and Gutnick, 1990
; Wang and McCormick, 1993
). After injection of depolarizing current pulses, these IB-like cells generated a group of two or more spikes riding on a depolarizing hump. A more pronounced burst was elicited by current pulses given from a hyperpolarized membrane potential (not shown). All IB-like cells were layer V pyramidal neurons (Wang and McCormick, 1993
). The complete absence of epileptiform activity even in long-term cultures was due to a well-differentiated population of inhibitory cell types (~1215% GAD mRNA expressing neurons) (Obst and Wahle, 1997
). In a parallel study we describe the properties of interneurons in OTCs, and found basket neurons with horizontal and with columnar axons which displayed typical fast-spiking properties and firing rates higher than those of pyramidal neurons (Klostermann and Wahle, 1999
).
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Activity in OTCs was generated by synaptic transmission. Application of the GABAA receptor antagonist bicuculline (0.1 mM, n = 10 cells) caused a disinhibition indicated by prolongated EPSPs and a hyperexcitability with paroxysmal depolarization shifts (PDSs) and epileptiform discharges (Fig. 2A.24). Removal of bicuculline restored the balance of excitation and inhibition (Fig. 2A.5
). The spontaneous activity decreased with increasing Mg2+ concentrations (washed in for 10 min; Fig. 2B.2,3
). In the cells tested (n = 48 cells), spontaneous action potentials were not observed under 7.5 and 10 mM Mg2+, although the neurons still generated action potentials upon current injection (inset in Fig 2B.3
). Application of 10 mM Mg2+ only slightly altered resting potential (±2 mV). In contrast, application of 0.1 mM TTX reversibly prevented spontaneous and current pulse-evoked action potentials (Fig. 2B.5
). After recovery in ACSF containing 2 mM Mg2+ for 30 min3 h, the normal firing patterns recurred (Fig. 2B.4
). Acute application of the non-NMDA receptor antagonist NBQX (1 mM, n = 10 cells) initially reduced (during wash-in, Fig. 2C.2
) and finally eliminated the SBA (Fig. 2C.3
), although the neurons still generated action potentials upon current injection (inset in Fig 2C.3
). Normal spike rates recurred after washout (Fig. 2C.4
). Baker et al. reported that a long-term blockade of action potential activity with selected glutamate receptor antagonists fails because neurons adapt to the treatment (Baker et al., 1998
). We therefore decided to chronically block the SBA with 10 mM Mg2+ added to the culture medium.
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Another 10 pyramidal cells from layers V/VI and II/III grown under chronic blockade for 3050 DIV were allowed to recover for either 3 DIV (n = 5) or 5 DIV (n = 5) in normal medium. As expected, all cells displayed SBA. Cells recorded after a 3 DIV recovery often had slightly elongated EPSPs (Fig. 3D). However, none displayed PDSs. After a 5 DIV recovery period, none of the cells had elongated EPSPs and none displayed epileptiform activity (Fig. 3E
). Hyperexcitability was thus no longer detected. However, 8/10 cells recorded in layers II/III and V/VI responded to current injections with an IB-like pattern. On current injections, they generated peculiar groups of two or three spikes (Fig. 3F
), and in most cells this effect was more pronounced after stimulation from a more hyperpolarized membrane potential (Fig. 3F2
). This IB-like pattern was found in all cells irrespective of laminar position. The other two neurons revealed the RS pattern.
Morphology of Pyramidal Cells
As representative examples, two pyramidal cells from normal OTCs aged 19 DIV (Fig. 4AC) and 73 DIV (Fig. 4DF
) and one pyramidal neuron aged 35 DIV from a chronically blocked OTC (Fig. 4GI
), are shown to demonstrate the high degree of structural maturation. These layer V neurons had apical dendritic tufts reaching into layer I and a bunch of basal dendrites. Dendritic side branches ended as tapering processes. We never found growth cone-like specializations at the tips of dendritic or axonal branches. Dendrites were covered by spines of normal morphology. Somatic or conspicously long dendritic appendages, usually interpreted as immature spines, had neither been observed in pyramidal cells from normal OTCs nor from age-matched activity-blocked OTCs. The 19 DIV cell was more densely covered with spines than the 73 DIV cell. As a first attempt towards statistical comparison, we have analyzed the spine densities of our reconstructed cells from normal and activity-blocked OTCs, and found such a high variability already between cells of virtually identical morphology, laminar position and age grown under the same experimental condition that we cannot tell whether the blockade or transient epileptiform activity has led to a numerical spine misdevelopment. The axons had very fine varicosities and ramified extensively thoughout the explant. Note, for instance, that the axonal domain of the 35 DIV cell was quite similar to the 19 DIV cell. All cells gave the impression of mature, well-differentiated neurons. Pyramidal neurons are the major source of neurotrophins in the neocortex in vivo, and we next analyzed the BDNF mRNA expression in vivo and in OTCs employing identical PCR conditions.
Developmental Expression of BDNF mRNA in OTC Compared to Visual Cortex in Vivo
In vivo, BDNF mRNA expression was low during the first two weeks (Fig. 5A). At postnatal day (P) 23 BDNF mRNA had dramatically increased. Peak levels were observed at P40. Thereafter the expression declined to adult values and then remained constant. The time course in OTC (Fig. 5B
) revealed an initial decrease at 5 DIV, most likely due to the trauma of explantation, but the expression recovered quickly, and by 10 DIV was already slightly higher than at P10 in vivo. The expression remained more or less at a plateau thereafter. In situ hybridization revealed BDNF mRNA expressing pyramidal neurons in all layers except layer I (Gorba and Wahle, 1999
). The visual input-dependent reduction in expression in layers IV/V (Capsoni et al., 1997
) fails to occur in OTCs (Gorba and Wahle, 1999
). Overall, however, the time course expression was surprisingly similar with respect to early onset and adult plateau levels.
BDNF mRNA Expression Depends on SBA
BDNF mRNA was dramatically reduced in chronically blocked OTC at 15 DIV and in 50 DIV compared to age-matched spontaneously active OTC (Fig. 6A,B, bars 14, note that in this figure the 15 DIV mean value was set to 1). Returning OTCs after chronic blockade from 314 DIV or from 330 DIV to normal medium for 30 DIV resulted in an increase of BDNF mRNA expression in OTC (Fig. 6A,B
, bars 5,6). A short-term recovery period of 16 h was sufficient to increase BDNF mRNA to levels observed in 15 and 50 DIV control OTCs (Fig. 6A,B
, bar 7). The reverse experiment was perfomed in OTC grown for 50 DIV as spontaneously active OTCs. After 5 DIV of blockade BDNF mRNA had declined to levels observed in chronically blocked OTCs (Fig. 6A,B
, bar 8). Already a 12 h period of activity blockade caused a marked reduction in expression (Fig. 6B
, bar 9). A period of 3 h, however, did not cause a detectable decrease. In contrast, when comparing the levels of NT-3, NT-4/5, and trkC and trkB receptor in spontaneously active versus chronically blocked OTC (Fig. 6C,D
, all at 50 DIV) no changes were observed. The level of these mRNAs was neither reduced nor upregulated.
Laminar-specific Recovery of BDNF Expression
Electrophysiology has revealed a period of hyperexcitability shortly after withdrawal of the Mg2+ blockade. Although we had expected a higher than normal level of BDNF expression, the PCR revealed that after short-term and long-term recovery BDNF mRNA expression has only increased to values observed in age-matched, spontaneously active cultures. This led us to analyze the period of short-term recovery more closely with in situ hybridization. Compared to active OTCs (Fig. 7A), only very few neurons had remained above detection limit in chronically blocked OTC (Fig. 7C
). After 3 h recovery of SBA a faint BDNF signal in the uppermost cortical layer was detected (Fig. 7D
). After 16 h of recovery, the upper layers II/III were very intensely labeled. Strikingly, signals were still absent from deeper cortical layers (Fig. 7E
), although the electrophysiology has revealed that neurons in all layers display action potential activity. After 3 and 5 DIV recovery the BDNF hybridization signal was homogeneously distributed in all layers and had a distribution and intensity very similar to control OTCs (Fig. 7F
). The same experiments were perfomed with OTCs that had been activity blocked for up to 60 DIV, and the BDNF mRNA expression recovered with the time course and laminar expression profile documented here for a set of 35 DIV OTCs. In contrast to this peculiar layer-specific pattern of recovery, an activity blockade for 24 h (performed with OTCs grown initially for 30 DIV as spontaneously active cultures) resulted in a homogeneous decline of BDNF mRNA in all layers (Fig. 7B
).
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Rapid BDNF mRNA Expression Recovery in Upper Layers Depends on Calcium Influx
Next, we asked which postsynaptic receptors or channels were mediating the rapid BDNF mRNA increase in supragranular layers during recovery of the SBA. BDNF mRNA expression is driven by glutamate binding to NMDA- and non-NMDA-receptors (Takahashi et al., 1993; Lindholm et al., 1994
), and calcium influx through voltage-gated channels (Shieh et al., 1998
; Tao et al., 1998
). Therefore, in 30 DIV cultures we antagonized kainate/AMPA and NMDA-type glutamate receptors, and L-type calcium channels for 16 h after the end of an activity blockade, and analyzed the effects on BDNF mRNA with reverse transcription (RT) PCR and in situ hybridization. As control, 330 DIV chronically blocked OTCs were treated with normal medium without addition of antagonists (Fig. 8A,B
, bar 2; Fig. 9A
), which confirmed the previous results (see Figs 6, 7
). The massive BDNF mRNA increase in supragranular layers observed after a 16 h recovery of SBA was neither prevented by the non-NMDA antagonist NBQX (Fig. 8A,B
, bar 3; Fig. 9B
) nor by the NMDA antagonist APV (Fig. 8A,B
, bar 4; Fig. 9C
). In contrast, a blockade of L-type Ca2+-channels with nifedipine resulted in a marked reduction of the BDNF mRNA upregulation (Fig. 8A,B
, bar 5; Fig. 9D
). The combination of nifedipine and APV completely prevented the upregulation, and expression remained at the level of chronically blocked cultures (Fig. 8A,B
, bar 6; Fig. 9E
).
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Discussion |
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The rat visual cortex is constituted by ~85% pyramidal cells and ~15% interneurons. All are still immature in the newborn (Miller, 1981; Meyer and Ferres-Torres, 1984
) and commence most dendritic and intracortical axonal growth and synaptogenesis after birth (Sutor and Luhmann, 1995
), and thus after explantation. Differentiated cell types are evident after two weeks in OTCs (Obst and Wahle, 1995
, 1997
; Kierstein et al., 1996
; Plenz and Aertsen, 1996
), and class-specific features have emerged. The layer V pyramidal cells presented have formed extensive apical tufts in layer I as do corticotectal cells (Wang and McCormick, 1993
; Cardoso de Oliveira and Hoffmann, 1995
). Apical dendrites of layer VI pyramidal cells ended in middle cortical layers (Obst and Wahle 1997
) (their Fig. 4
), suggesting that such class-specific features (Koester and O'Leary, 1992
) emerge in the absence of a target region. Typical morphological features were also observed in pyramidal cells grown in the absence of SBA. All had extensively branching axons and a wealth of intrinsic connections. Dantzker and Calloway found the laminar specificity of axonal branching to be slightly altered under activity blockade; axon termination, however, was unchanged (Dantzker and Calloway, 1998). Several structural proteins, including the synapse marker synaptophysin, display a normal time course of expression in activity-blocked OTC compared to normal ones and cortex in vivo (Kierstein et al., 1996
). Furthermore, the structural differentiation of interneurons is presumably not affected by activity blockade, because the morphology, axonal branching and perisomatic terminal patterns of parvalbuminergic basket neurons matches those from spontaneously active OTCs (T. Gorba et al., in preparation). Since targeting inhibitory and excitatory synapses is activity independent (Benson and Cohen, 1996
), it supports the assumption of a largely activity-independent differentiation of the synaptic network.
The physiological parameters of our cells are very similar compared to neurons recorded, for example, in acute slice preparations. A majority is regular spiking (RS), and only few layer V neurons had an IB-like pattern (Connors and Gutnick, 1990; Mason and Larkman, 1990
; McCormick et al., 1993
). As a major difference, spontaneous activity rates in OTCs are lower than in vivo. Although some neurons in OTCs displayed spike rates of up to 300 Hz upon current injections, only few displayed spontaneous activity rates close to the ranges reported in vivo [5.7 I/s (Herz et al., 1964
), 110 I/s (Creutzfeld et al., 1969
), up to 20 Hz (Kobayashi et al., 1993
)]. OTCs lack sensory input, and SBA arises only via the intrinsic synaptic connectivity. Therefore, pyramidal cells in OTCs display a ~10-fold lower level of SBA. Only the fast-spiking interneurons develop significantly higher rates (Klostermann and Wahle, 1999
). The molecular repertoire of neurotransmitter receptors could be assumed to develop normally in OTCs. We have so far screened by RT-PCR the expression of GLUR14, 5, 6, 7, the kainate receptor 2, NMDAR1, and GABAA receptor subunits ß13 and
, in normal OTCs, in activity-blocked OTCs and for comparison in the cortex in vivo. So far all analyzed receptors have also been detected in normal and in activity-blocked OTCs, although exact expression levels remain to be determined (Klostermann et al., 1996
). For instance, the presence of all GABAA receptor subunits analyzed suggests effective inhibitory mechanisms, which explains the low activity rates of pyramidal cells and especially the absence of pathological activity, which was seen only after pharmacological modifications or recovery after activity deprivation.
Effects of Activity Deprivation
After weeks of chronic blockade, pyramidal cells in all cortical layers instantly displayed synaptically driven action potentials. The ontogenetic expression of neurotransmitter receptors and intracellular mechanisms underlying generation of PSPs and action potentials must have proceeded in the absence of SBA. Excitability also appears immediately in microcultured neurons grown for weeks under high Mg2+ after return to ACSF (Segal and Fursphan, 1990). The presence of EPSPs, IPSPs, and the pharmacology indicate that glutamate is excitatory, while GABA has aquired its inhibitory role. The developmental reversal of the chloride gradient (Cherubini et al., 1991
) thus appears to occur independently of action potential activity. Only in neurons recorded during recovery from chronic blockade did we observe PDSs and prolongated EPSPs, which are clear symptoms of pathological, epileptiform activity (Segal and Fursphan, 1990
). However, after a 35 DIV recovery OTCs have managed to aquire de novo a well-balanced state of excitation and inhibition. Interestingly, the rather immature 14 DIV and the structurally well-differentiated 3040 DIV cortex were equally able to establish de novo a normal physiological working mode. The state is permanent, because any ongoing pathological activity would most likely have resulted in progressive cell loss; however, neuron numbers remain constant in OTC after longterm recovery (Wirth et al., 1998
).
Changes in firing properties have been observed. While control OTC had very few IB-like neurons (all in layer V), many pyramidal cells in all laminar positions displayed an IB-like mode after recovery from activity deprivation. A majority still fired IB-like after 5 DIV recovery when other symptoms of epileptiform activity were no longer detected. It remains to be determined what had caused the change in firing mode, and whether IB-like firing contributes to the epileptiform activity (Chagnac-Amitai and Connors, 1989; Connors and Gutnick, 1990
). For instance, RS neurons can switch to an IB mode depending on activation of certain receptors, dendritic depolarization, or calcium or potassium channel function (Friedman and Gutnick, 1989
; Connors and Gutnick, 1990
; McCormick et al., 1993
; Yuste et al., 1994
), the expression of which might have been impaired in activity-blocked OTC. Desai et al. recently found in early postnatal dissociated cortical neurons that activity deprivation dramatically increases the excitability of the neurons by upregulating sodium currents (and presumably channels) and downregulating a potassium current (Desai et al., 1999
). Together, the results suggest that the structural differentiation of a complex network can be dissociated from the physiological maturation. A cell's firing pattern depends on exogenous influences and it remains to be determined whether after long-term recovery most neurons would return to the more common RS-mode.
BDNF is Upregulated On Time in the Monocultured Cortex
Normal OTCs display a developmental increase of BDNF mRNA expression with a time course and adult plateau values similar to the cortex in vivo, although BDNF initially starts from a slightly depressed level due to the explantation. Sensory input is thus not required for the induction and maintenance of what appears to be an adult steady-state level. Rather, this working level is induced and maintained by the developing SBA via the intracortical circuitry. The activity of a few neurons in the network is sufficient to upregulate BDNF, because plateau values were reached during the second week when only about one-third of our recorded neurons displayed spontaneous action potentials. Expression is activated by glutamatergic transmission effective in the immature cortex, and subthreshold events thus most likely contribute to the developmental upregulation. Recent studies report a role of Ca influx and Ca-dependent kinases in the activity-dependent BDNF expression (Murray et al., 1998; Shieh et al., 1998
; Tao et al., 1998
). Calcium channels are present in the immature cortex (Galewski et al., 1992
), and CAM-kinases appear during the first postnatal week (Burgin et al., 1990
). Further, it was surprising that the increase in action potential rates and the much higher proportion of spontaneously active neurons in OTC older than 20 DIV did not elicit a further increase in BDNF expression. On the other hand, action potential rates, which in OTCs were ~10-fold lower than in vivo, maintain in old OTCs BDNF mRNA levels similar to the adult cortex in vivo. BDNF transcription thus does not follow the absolute activity levels. With regard to translation, it would be highly interesting to see whether higher activity in vivo elicits higher amounts of BDNF protein, since BDNF protein and mRNA may not correlate linearly due to post-transcriptional regulation (Nawa et al., 1995
).
The level of BDNF appears to be set by the balance between excitation and inhibition. Excitation matures earlier than inhibition (Sutor and Luhmann, 1995). The transient BDNF peak observed in vivo around P20 may thus be boosted by a higher excitability of the cortical network during the third postnatal week. A balanced state of excitation and inhibition, however, is reached by P20 in vivo (Sutor and Luhmann, 1995
), and thus could not be responsible for maintaining the BDNF peak until the end of the critical period [rat: P15-P45 (Fagiolini et al., 1994
)]. We observed the BDNF peak in vivo (Schoups et al., 1995
), but not in monocultured cortex. Also thalamocortical cocultures lacked a transient BDNF peak (own unpublished observation). This suggests that thalamic input alone is not sufficient to elicit the peak, and it is not occurring autonomously during early postnatal development. Rather, our data confirm the hypothesis that light and sensory experience after eye opening transiently sustain a higher BDNF expression in visual cortex during the critical period when BDNF may be required for plasticity (Castrén et al., 1992
; Maffei et al., 1992
; Lindholm et al., 1994
; Bozzi et al., 1995
).
BDNF Expression Depends on SBA
BDNF expression remains low in 14 DIV and in 50 DIV activity-blocked OTCs. There is no delayed upregulation in longterm activity-deprived OTCs. Rather, the onset of spike activity eliciting calcium influx into the postsynaptic neuron drives the BDNF mRNA expression (Shieh et al., 1998; Tao et al., 1998
). In our short-term recovery paradigm, SBA was observed immediately after return to normal ACSF, and first traces of BDNF mRNA were detected after 3 h. The level observed (with PCR) after 16 h recovery was in the range of the long-term recovery level. With regard to the transient period of epileptiform activity we expected higher values, because hyperexcitability in vivo dramatically upregulates BDNF (Nawa et al., 1995
). However, an upregulation caused by pathological activity in vivo sets off from the steady-state expression level, whereas in our culture paradigm it starts from a very low level. In fact, a few hours of recovery of SBA produced locally and transiently a dramatic overshoot of BDNF mRNA exclusively in supragranular layers. This resembles observations from kindling experiments (Metsis et al., 1993
; Kokaia et al., 1994
; Murray et al., 1998
) or after induction of spreading depression (Kokaia et al., 1993
). Since, on the other hand, BDNF mRNA (Bozzi et al., 1995
) and protein (Rossi et al., 1999
) is downregulated, especially in the upper layers of visual cortex after monocular deprivation, it appears as if the BDNF expression is very plastic in supragranular layers. Our in situ hybridizations were performed in 3060 DIV OTCs. By that time in vivo, the highest density of voltage-gated calcium channels and of NMDA receptors has already shifted to supragranular neurons (Galewski et al., 1992
; Catalano et al., 1997
), which explains the localized BDNF increase, and the much more sluggish response of infragranular neurons. The BDNF mRNA increase after an activity blockade depends on calcium influx through L-type calcium channels in synergy with NMDA receptors. Calcium influx through NMDA receptors leads only to a transient phosphorylation of the BDNF-regulating transcription factor CREB, whereas its phosphorylation is sustained by calcium influx through L-type channels (Tao et al., 1998
). Therefore antagonizing solely the NMDA receptors had no effect on BDNF mRNA upregulation after the activity blockade, whereas nifedipine alone caused a clear reduction. A coapplication of APV and nifedipine then reduces the calcium influx below a minimum theshold for the induction of BDNF mRNA transcription. NBQX could not prevent the BDNF mRNA increase, because it failed to suppress the SBA long-term (Baker et al., 1998
). Blocking non-NMDA receptor-mediated excitation could be even more difficult after an activity blockade, because the amplitude of AMPA-mediated currents has been shown to be strongly potentiated after activity deprivation (Turrigiano et al., 1998
).
Synaptic transmission and hyperexcitability are the first to recover as revealed by the electrophysiology, and both firing modes of pyramidal neurons equally promote the de novo BDNF expression. The newly produced BDNF might then contribute to the transient hyperexcitability by potentiating synaptic transmission and even eliciting epileptiform activity (Berninger and Poo, 1996; Carmignoto et al., 1997
; Scharfman, 1997
). On the other hand, activity deprivation causes neurons to increase their excitability (Desai et al., 1999
), and the quantal amplitude of excitatory synapses between pyramidal cells is increased when BDNF is low (Rutherford et al., 1998
). The increasing BDNF during recovery from activity blockade in turn would increase the quantal amplitude of synapses of pyramidal cells to interneurons this way recruiting inhibition to pyramidal cells (Rutherford et al., 1998
). Furthermore, under conditions of increased activity inhibitory synapses depress less than excitatory ones, which would also favor the de novo establishment of a stable balance between excitation and inhibition (Galaretta and Hestrin, 1998
; Varela et al., 1999
). Within 35 days the transient epileptiform activity has ceased, which suggests a sufficient recruitment of interneurons. These interneurons then would in turn negatively regulate BDNF release (and synthesis?) from pyramidal cells via GABAA receptor-mediated inhibition (Marty et al., 1996
). This most likely explains why after a 5 DIV recovery BDNF expression in all cortical layers has returned to control level. Together this supports the view that the balance between excitation and inhibition rather than the absolute level of SBA determines the BDNF level.
An important point is that BDNF expression does not become constitutive. The BDNF decrease upon blockade onset in old OTCs, and its increase upon withdrawal of the blockade at 14, 30 or 50 DIV always occurred very quickly. The fast decline kinetic confirms the reportedly short half-life of the mRNA (Castrén et al., 1998). Fast kinetics were also reported in the visual cortex in response to light, and they persist long after the end of the critical period (Castrén et al., 1992
; Bozzi et al., 1995
; Schoups et al., 1995
). Such activity dependence is required for a molecule that is presumed to stabilize firing rates in cortical networks.
In contrast, NT-3 mRNA is unchanged in silenced OTCs, and also in visual structures in vivo after retinal TTX injections (Lein et al., 1997). NT-4/5 appears to be not directly regulated by activity (Lindholm et al., 1994
), at least not by induction of long-term potentiation (Castrén et al., 1993
). TrkC and trkB remain expressed, and the receptors remain functional. NT-3 probably mediates the structural differentiation of pyramidal neurons in an activity-independent way (Ghosh and Greenberg, 1995
; Baker et al., 1998
), and the axodendritic transfer of NT-3, for instance, is activity-independent (von Bartheld et al., 1996
). NT-3 promotes calbindin expression (Collazo et al., 1992
), which remains constant in activity-blocked OTCs (own observations). Exogeneous NT-4/5 promotes neuropeptide Y (NPY) expression even in the absence of SBA (Wirth et al., 1998
). Whether NT-3, NT-4/5 and the receptors are upregulated during the period of epileptiform activity remains to be shown; trkB mRNA, for instance, increases during spreading depression in upper cortical layers (Kokaia et al., 1993
). BDNF probably plays no role in morphological differentiation, because even if it is synthetized from the drastically reduced amount of mRNA and released by some activity-independent mechanisms, it is not effective because it requires activity as a cofactor to promote structural (McAllister et al., 1996
) and molecular differentiation (Wirth et al., 1998
). Together, this indicates that synthesis of receptors and ligands is differentially regulated by pyramidal neurons.
Effects on Interneurons
The lack of BDNF in activity-deprived OTCs particularly affects the interneurons. They rarely express trkC, but express trkB, and they do not synthetize BDNF (Gorba and Wahle, 1999). They depend on BDNF produced and released by pyramidal cells (Carnahan and Nawa, 1995
; Rocamora et al., 1996
; Marty et al., 1997
). Although at least some neurotrophins are released constitutively from proximal dendrites and somata (Canossa et al., 1997
) which are contacted, for example, by basket cells, these interneurons could presumably not aquire sufficient trkB signaling to achieve proper neurochemical differentiation. NPY mRNA expression, for instance, is reduced in activity-blocked OTCs (Wirth et al., 1998
), while SBA and BDNF in the presence of SBA promote NPY expression (Carnahan and Nawa, 1995
; Nawa et al., 1995
; Obst and Wahle, 1997
; Wirth et al., 1998
). This could be a mechanism that helps to suppress epileptiform activity (Klapstein and Colmers, 1997
). Wirth et al. reported that despite recovery of SBA and recovery of BDNF (as shown now in the present study) the NPY mRNA expression remained low (Wirth et al., 1998
). This failure of both epigenetic factors in stimulating NPY expression has led us to suggest that a neuron's expression profile must be specified by additional mechanisms during the early period of interneuronal differention (Obst and Wahle, 1997
; Wirth et al., 1998
). Besides NPY, glutamic acid decarboxylase is activity dependent. However, the expression on mRNA and protein level quickly recovers after activity deprivation (manuscript in preparation), and effective GABA- ergic synaptic inhibition was observed in the acute recovery paradigm (see Results). The increase of action potential activity and of BDNF synthesis during recovery from activity deprivation thus recruits sufficient interneurons, promotes their final neurochemical differentiation and restores the balance of excitation and inhibition.
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