Cortical Spreading Depolarization (CSD) Recorded from Intact Skin, from Surface of Dura Mater or Cortex: Comparison with Intracortical Recordings in the Neocortex of Adult Rats.

In cerebral cortex of anesthetized rats single waves of spreading depolarization (CSD) were elicited by needle prick. CSD-related changes of DC (direct current) potentials were either recorded from the intact skin or together with concomitant changes of potassium concentration with K+-selective microelectrodes simultaneously at the surface of the dura mater or of the cortex ([K+]s) and in the extracellular space at a cortical depth of 1200 µm. At the intact skin CSD-related DC-shifts had amplitudes of less than 1 mV and had only in a minority of cases the typical CSD-like shape. In the majority these DC-shifts rose and recovered very slowly and were difficult to identify without further indicators. At dura surface CSD-related DC shifts were significantly smaller and rose and recovered slower than intracortically recorded CSD. Concomitant increases in [K+]s were delayed and reached maximal values of about 5 mM from a baseline of 3 mM. They rose and recovered slower than simultaneously recorded intracortical changes in extracellular potassium concentration ([K+]e) that were up to 65 mM. The results suggest that extracellular potassium during CSD is diffusing through the subarachnoid space and across the dura mater. In a few cases CSD was either absent at the dura or at a depth of 1200 µm. Even full blown CSDs in this cortical depth could remain without concomitant deflections at the dura. Our data confirmed in principle the possibility of non-invasive recordings of CSD-related DC-shifts. For a use in clinical routine sensitivity and specificity will have to be improved.

X-irradiation-induced changes in the diffusion parameters of the developing rat brain.

Three diffusion parameters of brain tissue, extracellular space volume fraction (alpha), tortuosity (lambda) and non-specific uptake (kappa') of tetramethylammonium were studied in the somatosensory neocortex and subcortical white matter of the rat during postnatal development (postnatal days 2-21) after X-irradiation at postnatal days 0-1. The diffusion parameters were determined from extracellular concentration-time profiles of tetramethylammonium. The tetramethylammonium concentration was measured in vivo with ion-selective microelectrodes positioned 130-200 microns from an iontophoretic source. X-irradiation with a single dose of 40 Gy resulted in typical early morphological changes in the tissue, namely cell death, DNA fragmentation, extensive neuronal loss, blood-brain barrier damage, activated macrophages, astrogliosis, increase in extracellular fibronectin and concomitant changes in all three diffusion parameters. The changes were observed as early as 48 h post-irradiation (at postnatal days 2-3) and still persisted at postnatal day 21. On the other hand, X-irradiation with a single dose of 20 Gy resulted in relatively light neuronal damage and loss, while blood-brain barrier damage, astrogliosis and changes in diffusion parameters were not significantly different from those found with 40 Gy. It is known that the volume fraction of the extracellular space in the non-irradiated cortex is large in newborn rats and diminishes with age [Lehmenkühler A. et al. (1993) Neuroscience 55, 339-351]. X-irradiation with a single dose of 40 or 20 Gy blocked the normal pattern of volume fraction decrease during postnatal development, and in fact brought about a significant increase. At postnatal days 4-5, alpha increased to 0.49 +/- 0.036 in layer III, 0.51 +/- 0.042 in layer IV, 0.48 +/- 0.02 in layer V, 0.48 +/- 0.028 in layer VI and 0.48 +/- 0.025 in the white matter. The large increase in alpha persisted three weeks after X-irradiation. Tortuosity and non-specific uptake decreased significantly at postnatal days 2-5; at days 8-9 they were not significantly different from those of control animals, while they increased significantly at days 10-21. Less pronounced but significant changes in all three diffusion parameters were also found in areas in the ipsilateral hemisphere adjacent to directly X-irradiated cortex. Compared to the control animals [Lehmenkühler A. et al. (1993) Neuroscience 55, 339-351], a significant decrease of alpha, lambda and kappa' was found in the contralateral hemisphere 48-72 h after X-irradiation. Later, alpha values were not significantly different from those in control animals. The decrease in lambda persisted at postnatal days 4-5. A significant increase in lambda and kappa' was found at postnatal days 18-21. We conclude that X-irradiation of the brain in the early postnatal period, even when it results in only relatively light damage, still produces changes in all three diffusion parameters, particularly a large increase in extracellular space volume fraction in all cortical layers, and in the subcortical white matter. Such changes in extracellular volume fraction of the brain can contribute to impairment of signal transmission, e.g. by diluting ions and neuroactive substances released from cells, and can play an important role in functional deficits, as well as in the impairment of developmental processes. Moreover, the increase in tortuosity (inferred from the decrease in apparent diffusion coefficient) in the X-irradiated cortex, as well as in the contralateral hemisphere, suggests that, even when extracellular volume is large, the diffusion of the substances is substantially hindered.

Extracellular space parameters in the rat neocortex and subcortical white matter during postnatal development determined by diffusion analysis.

Extracellular space volume fraction, tortuosity and nonspecific uptake of tetramethylammonium--three diffusion parameters of brain tissue--were measured in gray matter of the somatosensory neocortex and subcortical white matter of the rat during postnatal development. The three parameters were determined from concentration-time profiles of tetramethylammonium in postnatal days 2-120 in vivo. Tetramethylammonium concentration was measured with ion-selective microelectrodes positioned 130-200 microns from an iontophoretic source. Data were correlated with cytoarchitectonic structure and average thickness of the regions in 0-90-day-old rats using rapidly frozen tissue. Extracellular space volume fraction was largest in the newborn rats and diminished with age. In two-to three-day-old animals, volume fraction (mean +/- S.E.) was 0.36 +/- 0.04 in layers III and IV, 0.38 +/- 0.02 in layer V, 0.41 +/- 0.01 in layer VI and 0.46 +/- 0.01 in white matter. The earliest decrease in volume fraction was found in layers V and VI at postnatal days 6-7 followed by a decrease in layer III and IV at postnatal days 8-9 and in white matter at postnatal days 10-11. A further dramatic reduction in volume fraction occurred in all cortical layers and especially in the white matter between postnatal days 10 and 21. There was no further decrease in volume fraction between postnatal day 21 and adults (90-120 days old). The adult volume fraction values were: layer II, 0.19 +/- 0.002; III, 0.20 +/- 0.004; IV, 0.21 +/- 0.003; V, 0.22 +/- 0.003; VI, 0.23 +/- 0.007; white matter, 0.20 +/- 0.008. Values of tortuosity ranged between 1.51 and 1.65, nonspecific cellular uptake varied from 3.3 x 10(-3)/s to 6.3 x 10(-3)/s. The variations in each parameter were not statistically significant at any age. These data represent the first characterization of diffusion parameters in a developing brain. They confirm previous histological indications of a relatively large extracellular volume fraction during early postnatal development. The constancy of the tortuosity shows that diffusion of small molecules is no more hindered in the developing brain than in the adult. The large extracellular space volume fraction of the neonatal brain could significantly dilute ions, metabolites and neuroactive substances released from cells, relative to release in adults, and may be a factor in preventing anoxia, seizure and spreading depression in young animals. The diffusion characteristics could also play an important role in the developmental process itself.

The influence of the convulsant pentylenetetrazol on Ca2+-selective microelectrodes (neutral carrier ETH 1001).

Interference of the epileptogenic drug pentylenetetrazol (PTZ) on the Ca2+-selective microelectrode based on the neutral carrier ETH 1001 is described. It is suggested that tetraphenylborate, a component of the Ca2+-selective membrane, is responsible for this interference. Though the sensitivity of the Ca2+-selective microelectrode to PTZ is low, the error in measurements of the free Ca2+ concentration in biological preparations treated with PTZ has to be considered, since pathological changes in the extracellular Ca2+ concentration also produce relatively small changes in the relative electrode potential.

Continuous measurement of pentylenetetrazol concentration by a liquid ion exchanger microelectrode.

A double-barrelled microelectrode is described, which permits the continuous measurement of the concentration of the epileptogenic agent pentylenetetrazol (PTZ). The electrode is based on the liquid potassium exchanger (Corning No. 477 317) and enables measurements of PTZ concentration in physiological salines down to 1 mM. The electromotive behaviour of the liquid membrane against PTZ cannot directly be described by the Nicolsky-Eisenman formalism. It is suggested that specific interactions of the PTZ molecule with the Corning ligand are involved in the potential generating mechanisms.

Diffusion of penicillin in agar and cerebral cortex of the rat.

The diffusion of penicillin was studied in agar gel and the cerebral cortex of the rat using pressure microinjection and ion-selective microelectrodes selective to penicillin. From the agar measurements a free diffusion coefficient for penicillin of 3.52 +/- 0.08 (mean +/- S.E.M.) X 10(-6) cm2.s-1 for 37 degrees C was determined. The tortuosity value in the cortex was 1.62 +/- 0.03 (mean +/- S.E.M.) at the same temperature implying an apparent diffusion coefficient of 1.34 +/- 0.07 (mean +/- S.E.M.) x 10(-6) cm2.s-1. This tortuosity value means that penicillin diffuses in the cortex in a similar manner to other extracellular substances. These diffusion values clarify previous estimates and permit accurate evaluation of epilepsy models based on the application of penicillin.

Critical volume of rat cortex and extracellular threshold concentration for a pentylenetetrazol-induced epileptic focus.

The initiation of focal interictal epileptiform activity (FIEA) has been shown to depend on the activation of a sufficiently large volume of brain tissue. We estimated the size of this 'critical volume' for the convulsant pentylenetetrazol (PTZ) by analyzing the diffusion following its microinjection into rat motor cortex. PTZ concentration was monitored 100-200 microm away from the injection site with a PTZ-sensitive microelectrode. Diffusion analysis in 0.3% agar yielded the free diffusion coefficient D (8.50 +/- 0.15 X 10(-6) cm2 x s(-1) at 37 degrees C, median +/- S.E.M.). In brain tissue, diffusion was modified by extracellular volume fraction (alpha), tortuosity (lambda = (D/ADC)1/2; ADC = apparent diffusion coefficient) and non-specific uptake (k'). Using a value of 0.2 for alpha from previous studies, we found values of lambda = 1.61 +/- 0.01, k' = 3.37 +/- 0.15 X 10(-3) s(-1) and an injected volume U of 5.16 +/- 0.45 X 10(-10) l for pulses without FIEA, and lambda = 1.95 +/- 0.06, k' = 6.24 +/- 1.73 X 10(-3) s(-1) and U = 7.40 +/- 0.66 X 10(-10) l for pulses with FIEA. From the calculated concentration distribution of PTZ during FIEA we estimated a threshold concentration of about 1.77 mM PTZ and a volume with a radius of about 219 microm in which this concentration had to be exceeded. Since this critical volume was comparable in size to foci elicited by penicillin or electric stimuli in previous studies, it is concluded that it is determined by intrinsic tissue properties rather than by the convulsive agent being used.

Threshold extracellular concentration distribution of penicillin for generation of epileptic focus measured by diffusion analysis.

The tissue volume required to produce a penicillin-induced interictal discharge in the local EEG was estimated. A pair of microelectrodes were lowered into the motor cortex of anaesthetised and artificially ventilated rats. One double-barrelled electrode was used to release tetramethylammonium (TMA+) by iontophoresis or to pressure eject a solution containing penicillin (PEN-) and TMA+ concentration. The extracellular distribution of PEN- was defined using diffusion analysis of the TMA+. From these data the spatial distribution of PEN- was estimated at the times of first interictal spikes in the EEG. The critical mass of active nerve cells was calculated from the threshold concentration of PEN- needed to elicit paroxysmal depolarisation shifts in neocortical slices and found to lie within a tissue sphere with a radius of ca. 150 microns.

Diffusion analysis of valproate and trans-2-en-valproate in agar and in cerebral cortex of the rat.

The diffusion of valproate (VPA) and trans-2-en-valproate were studied in agar gel and in the cerebral cortex of the rat using pressure microejection and VPA-selective microelectrodes. From the agar measurements a free diffusion coefficient for VPA of 6.52 x 10(-6) cm2.s-1 and for trans-2-en-VPA of 5.25 x 10(-6) cm2.s-1 for 37 degrees C was determined. The tortuosity value in the cortex was 1.92 for VPA and 1.67 for trans-2-en-VPA. The tortuosity values suggest that VPA and trans-2-en-VPA diffuse mainly in the extracellular space of the brain.

Postnatal conditioning for spreading cortical depression in the rat brain.

The cerebral cortex of anaesthetised 2- to 12-day-old rats was superfused with artificial cerebrospinal fluid containing 100 mM acetate substituted for chloride to condition the brain for spreading depression (SD). After such superfusion, the earliest SD-like events were found at day 9 and full blown SD at day 10, whereas in the unconditioned brain the first SD occurred between days 12 and 15. Acetate conditioning of the cerebral cortex may be used to unmask neuronal and glial properties that are hidden in early stages of development.

Spreading depression can be restricted to distinct depths of the rat cerebral cortex.

In cerebral cortex of rats recurrent and single spreading depressions (SDs) were elicited by KCl application and by needle prick, respectively. SDs were monitored by recording changes of DC (direct current) potentials and of K+ concentration ([K+]o) in the extracellular space using K(+)-selective microelectrodes. Profiles of DC potential and of [K+]o were obtained by stepwise lowering a microelectrode array consisting of up to four electrodes into the brain cortex. Recurrent SDs propagating from the site of KCl application had lower frequencies and longer duration in superficial compared to deeper cortical structures. Single SD elicited by needle prick 3 mm away from the recording sites usually invaded the whole grey matter and showed DC potential shifts that differed in shape from the recurrent SDs. At a depth restricted to 1000 microns SD-related DC potential shifts and rises in [K+]o were drastically diminished. In 2 of 8 experiments prick-elicited SD was absent either above or below cortical depths of 800-1000 microns. The results suggest a barrier for vertical SD propagation in cortical depth between 800 and 1200 microns. The observations are relevant for application of noninvasive techniques (DC electroencephalography, magnetoencephalography) to detect SD in the brain.

Hypoxia- and hypercapnia-induced DC potential shifts in rat at the scalp and the skull are opposite in polarity to those at the cerebral cortex.

In anaesthetized and artificially ventilated rats DC (direct current) potential shifts induced by hypoxia or hypercapnia for 10 min were monitored at the surface of the skin, of the skull and of the cerebral cortex. Hypoxia was induced by decreasing the inspiratory O2 content from 20 to 10 or to 6% O2 in N2. Hypercapnia was induced by applying gas mixtures with CO2 contents from 0 to 5, 10, 20 or 30% CO2 in O2. DC potentials were recorded with non-polarizing electrodes filled with 150 mM NaCl solution. Hypercapnia evoked a negative DC shift (1.2-1.9 mV) epicranially and a large positive DC deflection (8.8-17.1 mV) epidurally. In contrast, hypoxia elicited a positive DC shift in skull recordings with a negative DC shift (2.6-3.1 mV) overshooting the baseline during recovery. DC shifts at the skin were positive, but smaller in amplitude (up to 3.1 mV). At the surface of the dura hypoxic DC shifts were negative and smaller (1.5-2.2 mV) than at the skull. The results show that a negative DC shift induced by hypercapnia recorded non-invasively from the skin or the skull is not always reflecting an increased cortical activation state and, vice versa, a positive DC shift does not always reflect a decreased state of neuronal activation. The electrogenesis of gas content-induced potentials could be due to the electrochemical diffusion of ions through the selective permeability barrier between the blood and tissue compartment. These observations are relevant for any application of non-invasive DC electroencephalography in the human cortex.

Extracellular changes of inorganic phosphate are different during spreading depression and global cerebral ischemia of rats.

Tissue levels of inorganic phosphate (iP-) and lactate (lac) increase during cerebral ischemia and cortical spreading depression (SD). Since cell membranes become leaky during these insults, iP- and lac were expected to leak into the extracellular space (ECS). In order to find out whether this occurs or does not, a microdialysis (MD) fiber was implanted into the cortex of anesthetized rats and extracellular lactate (lac(e)) and extracellular iP- (iPe-) were determined during various insults. Extracellular lactate increased to about the same extent during ischemia and SD. In contrast, iPe- increased during ischemia but not during SD. Instead, iPe- started to rise after SD and reached its maximum about 45 min later. The distinct pattern of iPe- in comparison to lac(e) during the above mentioned insults points to a qualitative difference of the underlying mechanisms: whereas lac appears within the ECS at any stressful situation, elevation of iP- within the ECS indicates depletion of energy stores in parallel to the lack of control of ion homeostasis.

Decrease of free calcium concentration at the outer surface of identified snail neurons during paroxysmal depolarization shifts.

Changes of free calcium concentration at the outer neuronal surface during paroxysmal depolarization shifts elicited by pentylenetetrazol were measured. Investigations were performed on the identified neuron B3 of the buccal ganglion of Helix pomatia. Extracellular calcium concentration was recorded by calcium-selective microelectrodes. The extracellular calcium concentration steeply decreased with the commencement of paroxysmal depolarization and started to reincrease when the paroxysmal depolarization had reached its plateau level. It is concluded that an influx of calcium ions takes place during paroxysmal depolarization shifts.

Caffeine-induced epileptic discharges in CA3 neurons of hippocampal slices of the guinea pig.

In order to analyze the elementary mechanisms underlying caffeine-induced epileptiform discharges, hippocampal slices of guinea pigs were exposed to this drug. When the bath concentration of caffeine exceeded 0.2 mM, periodically occurring paroxysmal depolarizations (PD) in CA3 neurons appeared. They were accompanied by declines of extracellular free calcium concentration and were suppressed by the organic calcium antagonists verapamil and flunarizine. PD-like fluctuations of the membrane potential could be evoked also in CA3 neurons functionally isolated by tetrodotoxin (TTX). The observations indicate that caffeine-induced PD are generated endogenously and that transmembranous calcium currents contribute to these mechanisms.