Chronic blockade of bioelectric activity in neonatal rat neocortex in vitro: physiological effects

We have examined what effect the loss of spontaneous bioelectric activity has on neural network formation in organotypic rat neocortical explants grown under serum-free culture conditions. Explants were taken from dorsal midline (presumptive visual) and lateral (presumptive auditory) occipital cortex and chronically exposed to tetrodotoxin which blocked all measurable bioelectric activity between change of medium. Extracellular recordings revealed complex, rhythmic spontaneous and evoked multiunit discharges in all explants examined (following tetrodotoxin washout in the experimental group). Control auditory explants had significantly more sites from which electric activity could be recorded compared with control visual explants. Auditory cultures showed no effect of the tetrodotoxin treatment, whereas visual explants showed significant increases over control values, equalling the auditory values. This increased level of spontaneous bioelectric activity was maintained for at least 10 days following transfer of the cultures to control growth medium. There was no significant difference between control visual and auditory explants regarding the number of sites from which evoked activity was seen. Nor did either cortex group show an effect of tetrodotoxin on the number of sites from which evoked activity was seen. The frequency with which spontaneous bioelectric discharges occurred per site increased with age in auditory vs visual cortex. These differences, however, were abolished in the tetrodotoxin-treated groups. It was concluded that neocortical explants which have experienced chronic suppression of spontaneous electric activity did not suffer deficits in neural network formation, though there is an effect on the incidence and frequency with which such activity is given.     

Elevated potassium prevents neuronal death but inhibits network formation in neocortical cultures

Chronic depolarization is inimical to neuronal growth and synaptogenesis so that spontaneous action potential generation appears to be required for the normal cytomorphological maturation of neocortical networks. The efficacy of 25 mM K in suppressing spontaneous bioelectric activity was monitored by extra- and intracellular recording from the explants. Intracellular recording from individual neurons showed that membrane potentials were reduced to ca -30 mV in potassium cultures but rapidly repolarized to ca -50 mV when returned to normal growth medium. Though action potentials could be readily evoked from these explants, spontaneous discharges and postsynaptic potentials were absent from potassium-treated cultures. Both spontaneous bioelectric activity and postsynaptic potentials returned to the cultures by 5 days after returning the explants to normal growth medium. Extracellular recordings also showed that the explants were bioelectrically silent in the presence of 25 mM K or 25 mM K plus tetrodotoxin. In contrast to tetrodotoxin alone, bioelectric activity was absent when the cultures (with or without tetrodotoxin) were returned to normal growth medium. The explants gradually began to evince spontaneous bioelectric activity between 3 and 5 days after being returned to normal growth medium. Massive cell death induced by chronic exposure to tetrodotoxin was totally prevented by concomitant addition of 25 mM potassium, though these explants were significantly thinner than controls due to a large decrease in neuropil. We conclude that chronic depolarization of neonatal cortical explants by potassium results in a delayed return of spontaneous bioelectric discharges. Chronic depolarization results in a retardation of network formation in these explants apparently due to a lack of neurite and/or synapse formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiological effects of sustained blockade of excitatory synaptic transmission on spontaneously active developing neuronal networks--an inquiry into the reciprocal linkage between intrinsic biorhythms and neuroplasticity in early ontogeny

Spontaneous bioelectric activity (SBA) taking the form of extracellularly recorded spike trains (SBA) has been quantitatively analyzed in organotypic neonatal rat visual cortex explants at different ages in vitro, and the effects investigated of both short- and long-term pharmacological suppression of glutamatergic synaptic transmission. In the presence of APV, a selective NMDA receptor blocker, 1-2- (but not 3-)week-old cultures recovered their previous SBA levels in a matter of hours, although in imitation of the acute effect of the GABAergic inhibitor picrotoxin (PTX), bursts of action potentials were abnormally short and intense. Cultures treated either overnight or chronically for 1-3 weeks with APV, the AMPA/kainate receptor blocker DNQX, or a combination of the two were found to display very different abnormalities in their firing patterns. NMDA receptor blockade for 3 weeks produced the most severe deviations from control SBA, consisting of greatly prolonged and intensified burst firing with a strong tendency to be broken up into trains of shorter spike clusters. This pattern was most closely approximated by acute GABAergic disinhibition in cultures of the same age, but this latter treatment also differed in several respects from the chronic-APV effect. In 2-week-old explants, in contrast, it was the APV+DNQX treated group which showed the most exaggerated spike bursts. Functional maturation of neocortical networks, therefore, may specifically require NMDA receptor activation (not merely a high level of neuronal firing) which initially is driven by endogenous rather than afferent evoked bioelectric activity. Putative cellular mechanisms are discussed in the context of a thorough review of the extensive but scattered literature relating activity-dependent brain development to spontaneous neuronal firing patterns.     

Growth of pyramidal, but not non‐pyramidal, dendrites in long‐term organotypic explants of neonatal rat neocortex chronically exposed to neurotrophin‐3

The present study was undertaken to determine the effects of neurotrophin-3 (NT3) and spontaneous bioelectric activity (SBA) on dendritic elongation and branching in long-term isolated organotypic explants of rat neocortex. Viral vector-directed expression of NT3 was used as an effective means to ensure a continuous, local production of the neurotrophic factor. Quantitative light microscopic measurement of dendritic branching patterns was carried out on Golgi-stained materials. Explants were exposed to an adenoviral vector encoding the genetic sequence for neurotrophin-3 (Ad-NT3), or to exogenous additions of the neuropeptide NT3. In order to test for activity-dependent growth effects under control and experimental conditions, explants were exposed to glutamatergic blockade using a cocktail of APV and DNQX. Both Ad-NT3 and NT3 peptide potently promoted apical and basal dendritic growth (elongation and branching) in pyramidal neurons. This growth was observed to be significant in layers II–IV and V. These growth effects were also not activity dependent, inasmuch as they were elicited from explants in which spontaneous bioelectric activity had been suppressed. Non-pyramidal neurons, throughout the neocortical slice, showed no significant dendritic responses to the prolonged presence of NT3. These findings show that pyramidal dendritic growth in long-term neocortical explants responds to at least one neurotrophic growth factor, NT3, and is independent of intrinsic bioelectric activity. The use of viral vectors in delivering a continuous high level of neurotrophic factor within developing neural tissues demonstrates its potential application to in vivo tissues during development, or in the stimulation of neuritogenesis and neuroregeneration following injuries.     

Chronic blockade of bioelectric activity in neonatal rat cortex grown in vitro: morphological effects

Culture thickness, numerical density of neurons and neuronal survival were studied in timed series of control and tetrodotoxin-silenced neocortical cultures to provide information on the role of bioelectric activity on neuronal development. In control cultures, culture thickness and number of surviving neurons decrease during the first weeks in vitro, but remain constant between 2 and 3 weeks indicating that the cultures are essentially mature. In the 4th week in vitro a further decrease in surviving neurons was observed. In tetrodotoxin-treated cultures the number of surviving neurons decreased significantly between 1 and 2 weeks in vitro, to remain constant thereafter. However, culture thickness significantly increased at 3 and 4 weeks in vitro after an initial drop between 1 and 2 weeks. Compared to age-matched controls at 2 and 3 weeks in vitro, only ca 50% of the neurons survived the loss of bioelectric activity. Similar differences were present between 1 and 2 weeks. Thus, the loss of all measurable bioelectric activity induces neuronal death in neocortical explants, but promotes neuropil formation by the surviving cells.     

A developmental decrease in NMDA-mediated spontaneous firing in cultured rat cerebral cortex

In primary cultures of fetal rat cerebral cortex chronic manipulation of the level and/or pattern of bioelectric activity leads to plastic changes in bioelectric activity, opposite to those seen during the manipulation. This suggests the presence of adaptive mechanisms which regulate functional development in the neuronal network. Since NMDA receptors play an important role in early postnatal bioelectric activity and have been implicated in activity-dependent plasticity in vivo, the involvement of NMDA and non-NMDA receptors in spontaneously occurring bioelectric activity was investigated in cultured rat cerebral cortex by assaying the effects of NMDA and non-NMDA antagonists on neuronal firing. In addition, the physiological consequences of chronic suppression of bioelectric activity were investigated following development in the presence of tetrodotoxin. NMDA receptors appeared at all ages to be more crucial for spontaneous bioelectric activity than non-NMDA receptors, although their relative importance decreased during the first 3 weeks. Whereas the NMDA antagonist APV strongly reduced burst firing, the non-NMDA antagonist DNQX tended to increase burst firing slightly. Following chronic suppression of bioelectric activity, non-variable burst firing was increased, thus replicating previous findings in cerebral cortex culture grown under different conditions. The prominence of NMDA receptor activation in spontaneous bioelectric activity in early cultures suggests a role for these receptors in activity-dependent functional plasticity, as found in vivo.     

Horseradish peroxidase tracing of dorsal root ganglion afferents within fetal mouse spinal cord explants chronically exposed to tetrodotoxin

Afferent projection patterns within organotypic explants of fetal mouse spinal cord-dorsal root ganglia (DRG) were mapped out histologically using an HRP-staining method. The role of spontaneous bioelectric activity in the development of cord-DRG connections was studied using tetrodotoxin (TTX) to eliminate action potential discharges in the experimental cultures. Cultures grown in TTX-supplemented medium had a significantly lower proportion of DRG afferents within the dorsal half of the cord explant than did untreated cultures. In addition, afferent fiber entrances were made predominantly on the dorsolateral aspect in control cord explants, in contrast with the more diffuse entrance pattern displayed by the experimental group. Comparison of those cultures in both groups where the fiber entrances were chiefly dorsal revealed a greater tendency in the TTX group for DRG afferents to grow ventrally after penetrating the cord. Thus, it appears that bioelectric activity of the target neurons may be required by DRG afferents for the development of selective innervation patterns.     

Homeostatically regulated spontaneous neuronal discharges protect developing cerebral cortex networks from becoming hyperactive following prolonged blockade of excitatory synaptic receptors

In order to further examine the role of spontaneous action potential (SAP) discharges in neocortical development, amino-acid-mediated synaptic transmission was selectively blocked in an improved organotypic neocortex culture preparation. Contralateral occipital cortex slices from neonatal rats were co-cultured for several weeks in a ventricle-to-ventricle orientation known to greatly enhance cyto-morphological and electrophysiological maturation. Such preparations are highly resistant to attempts to suppress neuronal firing by blocking ionotropic glutamate receptors: not only can kainate receptors partly substitute for NMDA- and AMPA-mediated neurotransmission when these receptors are pharmacologically blocked, but (muscarinic) cholinergic receptors also begin to drive SAP activity when the kainate receptors, too, are chronically blocked. Only tetrodotoxin proved able to eliminate SAPs altogether in these co-cultures, while GABAergic receptor blockade (using bicucculine) led to persistent epileptiform discharges. Treatment effects were assayed upon transfer to control medium by means of a quantitative analysis of spontaneously occurring polyneuronal spike trains. Total suppression of action potentials for several weeks (by tetrodotoxin treatment) led, as in earlier experiments, to strongly intensified burst firing upon transfer to control medium. Chronic glutamate receptor blocked cultures, on the other hand, showed only minor deviations from control firing levels and patterns when assayed in normal medium. Protection against the development of hyperactivity despite partial blockade of synaptic transmission was roughly proportional to the degree to which spontaneous firing during the treatment period approximated normal SAP levels. This homeostatic response to chronically reduced excitatory drive thus differs from earlier results using isolated organotypic cortex cultures, in which the restoration of SAP activity failed to prevent the development of network hyperactivity. Chronic bicucculine treatment, in contrast, had little or no homeostatic effect on SAP firing patterns; on the contrary, opposite to earlier findings using isolated occipital cortex explants, paroxysmal discharges persisted even after transfer to control medium.     

Effects of spontaneous bioelectric activity and gangliosides on cell survival in vitro

Chronic suppression of spontaneous bioelectric activity in spinal cord explants in the presence of tetrodotoxin (TTX) during network formation caused a large reduction in cell number (lowered DNA levels). The addition of gangliosides failed to protect against this cell loss. Conversely, the omission of galactose from the growth medium had no effect on DNA levels. It was concluded that the presence or absence of afferent selectivity is unlikely to require the survival of a regionally specific subpopulation of preferred dorsal root ganglion target cells. Neocortical explants also showed a large reduction in DNA levels following chronic TTX treatment, and morphometric analysis confirmed that neuronal survival was affected to the same degree. Chronic ganglioside supplementation failed to influence DNA and cell counts in either control or TTX-treated explants, but one of the added gangliosides (GD1a) stimulated extensive neuritic outgrowth in electrically silenced cultures. Particular ganglioside species, therefore, may exert a growth stimulating influence that can partially compensate for the absence of bioelectric self-stimulation during early development.     

Spatio-temporal analyses of stimulus-evoked and spontaneous stochastic neural activity observed by optical imaging in guinea pig auditory cortex

Stimulus-evoked response in the cortex involves random neural activity besides the deterministic responses reproducible to the stimulus. Recently, we have developed a new bright optical system that enables us to investigate the spatio-temporal patterns of such stochastic activity in the guinea pig auditory cortex without averaging. We show that (1) the stochastic neural activity is evoked by a tone-stimulus in addition to the deterministic response, and spontaneous stochastic activity is also observed in a similar manner; (2) our statistical estimation of optical responses such as variance showed that the evoked stochastic activity was increased by the sound stimulus compared to the spontaneous activity; (3) both types of stochastic activity mainly display oscillatory behavior, in the frequency range of 5-11 Hz; (4) there are no significant differences between the stimulus-induced and spontaneous stochastic neural activity in our statistical analyses using the PSD (power-spectrum density) and the spatial correlation function; (5) the spatial area of the evoked stochastic activity is not strongly correlated with the tonotopical area of the deterministic response that is mainly localized in the caudal area of field A of the guinea pig auditory cortex. Thus, the stochastic neural activity existing in the stimulus response and the spontaneous activity in the auditory cortex are possibly generated by a common neural mechanism. These results were confirmed statistically using 27 animals.     

Some functional effects of suppressing bioelectric activity in fetal mouse spinal cord-dorsal root ganglion explants

Organotypic explants of fetal mouse spinal cord-dorsal root ganglia were grown for 3 weeks in the presence of 10 mM magnesium ion, which effectively eliminated all recordable bioelectric activity throughout the culturing period. When tested in minimal essential medium, the chronically silenced explants had significantly fewer points from which spontaneous neuronal activity could be recorded. In addition, fewer points could be found that showed dorsal root ganglion-evoked responses, resulting from a greater tendency for the spinal cord activity to be restricted to the vicinity of the dorsally entering DRG fibers. These findings, therefore, support the hypothesis that spontaneous bioelectric activity is required for functional as well as structural maturation of neural networks.     

Impaired neurovascular coupling by transhemispheric diaschisis in rat cerebral cortex.

In acute brain disorders, elimination of the excitatory output from an injured brain region reduces activity in connecting brain regions remote from the lesion site (i.e., diaschisis). The authors examined the effect of functional ablation of the left cerebral cortex by cortical spreading depression (CSD) or topical application of tetrodotoxin on single cell spiking activity, baseline CBF, and neurovascular coupling in the right rat sensory cortex. CSD or tetrodotoxin in left cortex reduced the right cortical spontaneous spike rate by 36% and 45%, respectively. Baseline CBF in the right cortex was unaffected by a left-sided CSD, but decreased by 12% for left cortical application of tetrodotoxin. This suggested dissociation between spontaneous spiking activity and basal CBF. Left in-fraorbital nerve stimulation evoked local field potentials in right cerebral cortex that were reduced in amplitude by 19% for left CSD and by 23% for left tetrodotoxin application. The corresponding declines in the evoked CBF responses were 42% for CSD and 23% for tetrodotoxin. Vascular reactivity to adenosine remained unchanged in right cortex. Thus, transhemispheric diaschisis produced a pronounced decrease in the spontaneous spike rate accompanied by no reduction or a small reduction in basal CBF, and an attenuation in amplitudes of evoked synaptic responses and corresponding rises in CBF. The findings suggest that disturbed neurovascular coupling may contribute to the disturbance in brain function in acute transhemispheric diaschisis.     

Activity-dependent plasticity of inhibitory and excitatory amino acid transmitter systems in cultured rat cerebral cortex

Chronic suppression of spontaneous bioelectric activity in cultures of dissociated fetal rat cerebral cortex increases neuronal cell death and results in electrophysiological changes which indicate an altered balance between excitatory and inhibitory neurotransmission in culture. To delineate whether alterations in neurotransmitter release could underlie this imbalance, we investigated the effects of chronic tetrodotoxin (TTX) treatment on the content and release of glutamate, aspartate and γ-aminobutyric acid (GABA) in culture. Chronic TTX treatment decreased the content of all amino acids investigated. However, only GABA was decreased relative to the neuronal marker NSE (neuron-specific enolase), indicating a disproportionate loss of GABA production following chronic silencing. Depolarization-induced release of GABA, glutamate and aspartate increased about 10-fold between 7 and 21 days in control cultures. Chronic TTX treatment significantly increased the depolarization-induced release of glutamate and aspartate at 7 days in vitro relative to control levels. At all ages it caused a two-fold increase in the ratio of evoked excitatory amino acid release to that of GABA. These observations suggest that chronic silencing of developing neocortex cell cultures increases the ratio of excitatory to inhibitory synaptic activity either by differential cell death or by reduced synaptic efficiency, on which a decrease in GABA neurotransmission appears to play a major role. Since similar mechanisms may be involved in activity-dependent plasticity in vivo, these cultures provide a useful model to analyse this phenomenon at the cell biological and molecular level.     

Tetrodotoxin prevents posttraumatic epileptogenesis in rats.

Severe cortical trauma frequently causes epilepsy that develops after a long latency. We hypothesized that plastic changes in excitability during this latent period might be initiated or sustained by the level of neuronal activity in the injured cortex. We therefore studied effects of action potential blockade by application of tetrodotoxin (TTX) to areas of cortical injury in a model of chronic epileptogenesis. Partially isolated islands of sensorimotor cortex were made in 28- to 30-day-old male Sprague-Dawley rats and thin sheets of Elvax polymer containing TTX or control vehicle were implanted over lesions. Ten to 15 days later neocortical slices were obtained through isolates for electrophysiological studies. Slices from all animals (n = 12) with lesions contacted by control-Elvax (58% of 36 slices) exhibited evoked epileptiform field potentials, and those from 4 rats had spontaneous epileptiform events. Only 2 of 11 lesioned animals and 6% of slices from cortex exposed to TTX in vivo exhibited evoked epileptiform potentials, and no spontaneous epileptiform events were observed. There was no evidence of residual TTX during recordings. TTX-Elvax was ineffective in reversing epileptogenesis when implanted 11 days after cortical injury. These data suggest that development of antiepileptogenic drugs for humans may be possible.     

Physiological consequences of selective suppression of synaptic transmission in developing cerebral cortical networks in vitro: Differential effects on intrinsically generated bioelectric discharges in a living ‘model’ system for slow-wave sleep activity

Within the context of an updated thorough review of the literature concerning activity-dependent cerebro-cortical development, a survey is made of recent experiments which utilize spontaneous spike-trains as the dependent variable in rodent neocortex cultures when synaptic transmission is interfered with during early ontogeny. Emphasis is placed on the complexity of homeostatic adaptations to reduced as well as intensified firing. Two kinds of adaptation are distinguished: (i) rapid recovery (within several hours) towards baseline levels despite sustained blockade of excitatory synaptic transmission, and (ii) the generation of essentially normal firing patterns in cultures assayed in control medium following development in the presence of excitatory receptor blockers. The former category of homeostatic responses is strongly dependent on the type of preparation, with isolated organotypic explants showing greatly limited plasticity in comparison with co-cultures of matching contralateral pieces of cortical tissue. In such co-cultures, compensatory excitatory drive manifests itself even when all three known types of ionotropic glutamate receptors are chronically blocked, and is then mediated by (muscarinic) cholinergic mechanisms which normally do not contribute measurably to spontaneous activity.The rapid return of high levels of spontaneous firing during sustained selective glutamatergic receptor blockade appears to protect neuronal cultures treated in this way from becoming hyperexcitable. In particular, quasi-epileptiform paroxysmal bursting upon return to control medium, such as appears in preparations where bioelectric activity has been totally suppressed during network formation, fails to appear in chronically receptor blocked cultures. On the contrary, desensitization of blocked glutamate receptors, as a physiological compensation for the up-regulation of non-blocked receptors, could be demonstrated for both the AMPA and the NMDA glutamate receptor sub-types. This wide range of homeostatic responses underscores the importance of spontaneous neuronal discharges for setting and maintaining an optimal balance between excitatory and inhibitory mechanisms in developing neocortical networks.     

Physiological consequences of selective suppression of synaptic transmission in developing cerebral cortical networks in vitro: differential effects on intrinsically generated bioelectric discharges in a living 'model' system for slow-wave sleep activity

Within the context of an updated thorough review of the literature concerning activity-dependent cerebro-cortical development, a survey is made of recent experiments which utilize spontaneous spike-trains as the dependent variable in rodent neocortex cultures when synaptic transmission is interfered with during early ontogeny. Emphasis is placed on the complexity of homeostatic adaptations to reduced as well as intensified firing. Two kinds of adaptation are distinguished: (i) rapid recovery (within several hours) towards baseline levels despite sustained blockade of excitatory synaptic transmission, and (ii) the generation of essentially normal firing patterns in cultures assayed in control medium following development in the presence of excitatory receptor blockers. The former category of homeostatic responses is strongly dependent on the type of preparation, with isolated organotypic explants showing greatly limited plasticity in comparison with co-cultures of matching contralateral pieces of cortical tissue. In such co-cultures, compensatory excitatory drive manifests itself even when all three known types of ionotropic glutamate receptors are chronically blocked, and is then mediated by (muscarinic) cholinergic mechanisms which normally do not contribute measurably to spontaneous activity. The rapid return of high levels of spontaneous firing during sustained selective glutamatergic receptor blockade appears to protect neuronal cultures treated in this way from becoming hyperexcitable. In particular, quasi-epileptiform paroxysmal bursting upon return to control medium, such as appears in preparations where bioelectric activity has been totally suppressed during network formation, fails to appear in chronically receptor blocked cultures. On the contrary, desensitization of blocked glutamate receptors, as a physiological compensation for the up-regulation of non-blocked receptors, could be demonstrated for both the AMPA and the NMDA glutamate receptor sub-types. This wide range of homeostatic responses underscores the importance of spontaneous neuronal discharges for setting and maintaining an optimal balance between excitatory and inhibitory mechanisms in developing neocortical networks.     

Accelerated neural network formation in rat cerebral cortex cultures chronically disinhibited with picrotoxin

Our aim was to determine if chronic blockade of GABAergic inhibitory synaptic activity, monitored electrophysiologically at the neuronal level, would affect synapse formation and ultrastructure in dissociated fetal rat cerebral cortex cultures. This was achieved by adding picrotoxin to the serum-free growth medium in a dose that induced continuous epileptiform discharges throughout the culture period. Light and electron microscopic analysis suggested an accelerated synaptic network formation in the experimental cultures during the first 2 weeks in vitro. The elimination of excess synapses (mainly on spines), which normally takes place during the fourth week in vitro, occurred 1 week earlier in the presence of picrotoxin. Finally, the experimental cultures showed smaller spine synapses throughout the entire culture period. Because these effects were opposite those induced by chronic tetrodotoxin-blockade of spontaneous bioelectric activity in a previous study, the underlying causal factor could be the respective intensification and suppression of neuronal activity in the two experiments. An appropriate balance between excitatory and inhibitory synaptic drive seems therefore to be important for normal maturation of neocortical circuitry.     

Indications for a critical period for synapse elimination in developing rat cerebral cortex cultures

It was observed in an earlier study that chronic tetrodotoxin (TTX) blockade of spontaneous bioelectric activity (SBA) in rat cerebral cortex cultures prevented the large-scale elimination of synapses which normally occurs during the fourth week in vitro. This prompted us to study whether the persisting high synapse density during long-term TTX-treatment would still return to the ‘normal’ low level after restoration of SBA. Therefore, cultures grown in TTX-supplemented medium for 5 weeks were switched to control medium for an additional week prior to fixation. Electron microscopic analysis showed that the numerical synapse density remained at a high level, thus suggesting the presence of a critical period whereafter bioelectrically controlled elimination of redundant connections no longer occurs. In contrast, the mean size of synaptic structures depended only on the functional state of the tissue at the moment of fixation, being larger in TTX-silenced cultures than in bioelectrically active ones regardless of treatment during the first 5 weeks in vitro.     

Synaptogenesis in rat cerebral cortex cultures is affected during chronic blockade of spontaneous bioelectric activity by tetrodotoxin

Reaggregated occipital cortex cells of 19-day-old fetal rats were grown in a serum-free, chemically defined medium, and chronically exposed to impulse-blocking levels of tetrodotoxin (TTX) in order to study the role of bioelectric activity in synaptogenesis. As judged by phase-contrast microscopy, no differences were noticed in the development of neuronal networks in the TTX-treated vs control cultures. In addition, when TTX was withdrawn from experimental cultures at any stage of development, bioelectric activity qualitatively comparable to that of the control cultures appeared within 1 min. However, quantitative stereological EM analysis revealed a significant retardation in synapse formation and ultrastructural maturation of synaptic junctions during the first 3 weeks. Around 23 days in vitro, the central zone of the reaggregates in control cultures started to degenerate, but not earlier then day 27 in TTX-treated cultures. During this time, the control, but not the experimental cultures showed (in intact tissue regions mainly situated at the outside of the aggregates) a large and selective loss of spine synapses. It is concluded that functional blockade not only retards the early growth and maturation of synaptic networks but also prevents the later occurring selective loss of spine synapses.     

Cortical deafferentation in cat focal ischemia: disturbance and recovery of sensory functions in cortical areas with different degrees of cerebral blood flow reduction

During and after 15-min occlusion of the middle cerebral artery (MCA) in cats, local CBF and neuronal activity were measured in cortical areas varying in the degree of CBF reduction. In an area within the ischemic center (primary auditory cortex, middle ectosylvian gyrus), CBF was severely suppressed. Click-induced auditory evoked potentials and evoked as well as spontaneous single-unit activity ceased within 1 min after occlusion. Recirculation resulted in a recovery of the different neurophysiological parameters with a time delay ranging from several minutes to 2 h. In two areas surrounding the ischemic focus (a visual area in the marginal gyrus and the forelimb representation area in the primary somatosensory cortex), CBF was reduced but remained above 30 ml/100 g/min during MCA occlusion. Visual flash-induced evoked potentials and somatosensory evoked potentials induced by median nerve electrical stimulation ceased in the corresponding areas with a somewhat slower time course as compared to the auditory responses and they recovered faster after recirculation. In another somatosensory area (hindlimb projection area in the primary somatosensory cortex), CBF stayed nearly at control levels during occlusion. Evoked potentials and single-unit activity induced by tibial nerve electrical stimulation decreased approximately 5 min after occlusion and were abolished approximately 5 min later. At that time, single-unit activity had changed to a nonresponsive pattern but persisted. However, potentials evoked transcallosally by electrical stimulation of the contralateral hemisphere were still recorded. After reopening the MCA, the recovery of neuronal functions was usually complete and occurred within approximately 5 min.(ABSTRACT TRUNCATED AT 250 WORDS)