Untreated Cortex Research Articles

The Functional Significance of Gap Junction Channels in the Epileptogenicity and Seizure Susceptibility of Juvenile Rats

The functional significance of gap-junction (GJ) channels in seizure susceptibility and induction and maintenance of seizures in the developing rat brain was investigated on the 4-aminopyridine (4-AP) in vivo epilepsy model. In electrophysiological experiments, GJs were manipulated with a blocker or opener before induction or at the active epileptic foci between postnatal days 9 and 28 (P9-28). Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) amplification was used to measure the levels of connexin (Cx) 26, 32, 36, and 43 mRNAs at the untreated cortex or epileptic foci. The basic electrocorticogram (ECoG) and Cx messenger RNA (mRNA) expression patterns exhibited characteristic maturation; the 4-AP-induced epileptiform activity correlated well with these changes. Cx mRNA expressions were significantly upregulated around P16 (except for Cx26). The Cx26, 36, and 43 gene inducibility was highest around P16 and then declined significantly. In the youngest animals, the GJ opener induced rhythmic synchronous cortical activity. On maturation, the seizures became focalized and periodic; the discharges accelerated their amplitude and frequency increase. A transient decrease (P13-14) and then increase (P15-16) in seizure susceptibility were followed by a tendency to periodicity and focalization. The study suggests that GJ communication is involved in rhythm genesis and synchronization of cortical activity and may enhance the epileptogenicity of the developing brain.

Caspase Inhibitor z-DEVD-fmk Attenuates Calpain and Necrotic Cell Death in Vitro and after Traumatic Brain Injury

In studies designed to evaluate the therapeutic window for treatment of traumatic brain injury, the caspase 3 inhibitor z-DEVD-fmk improved neurologic function and reduced lesion volumes when administered at 1 but not at 4, 8, or 24 hours after injury. Moreover, neither caspase 3 nor PARP, a caspase 3 substrate, were cleaved in injured, untreated cortex from 1 to 72 hours after injury. Few cortical neurons expressed active caspase 3 or were TUNEL positive from 6 to 24 hours after injury, and TUNEL staining was primarily Type I (necrotic). Nissl staining revealed extensive neuronal necrosis in the injured cortex from 6 to 24 hours after impact. Considered together, these data suggested that z-DEVD-fmk may reduce neuronal necrosis, so we used an in vitro model of necrotic cell death induced by maitotoxin to test this further and explore the potential mechanism(s) involved. Z-DEVD-fmk (1 nM-100 microM) significantly attenuated maitotoxin induced neuronal cell death and markedly reduced expression of the 145 kD calpain-mediated alpha-spectrin breakdown product after maitotoxin injury. Neither the 120 kD caspase-mediated alpha-spectrin cleavage product nor cathepsin B were expressed after maitotoxin injury. In a cell free assay, z-DEVD-fmk reduced hydrolysis of casein by purified calpain I. Finally, z-DEVD-fmk reduced expression of the 145 kD calpain-mediated alpha-spectrin cleavage fragment after traumatic brain injury in vivo. These data suggest that neuroprotection by z-DEVD-fmk may, in part, reflect inhibition of calpain-related necrotic cell death.

Blockade of voltage-gated calcium channels in rat inhibits repetitive cortical spreading depression

Blockers of L-, N-, and P/Q-type voltage-gated calcium channels (VGCCs) were topically applied to the cortical surface of anaesthetized adult rats to study their role in cortical spreading depression (CSD), a correlate of the migraine aura. By pricking the brain, single CSD could still be elicited after blockade of the three different types of VGCCs as in the untreated brain. Topical KCl application to the untreated cortex resulted in repetitive CSD. However, after application of blockers at either L-, or N-, or P/Q-type VGCCs to the cortical surface, application of KCl elicited only one or very few CSD, and their repetition rate was dramatically reduced. The results suggest that cortical excitability resulting in repetitive CSD is markedly influenced by N- and P/Q-type VGCCs and less by L-type VGCCs.

Horizontal spread of synchronized activity in neocortex and its control by GABA-mediated inhibition

1. Suppression of GABAA receptor-mediated inhibition disrupts the neural activity of neocortex and can lead to synchronized discharges that mimic those of partial epilepsy. We have studied the role of GABAA-mediated inhibition in controlling the synchronization and horizontal (tangential) spread of cortical activity. 2. Slices of rat SmI were maintained in vitro and focally stimulated in layer VI while recording with a horizontal array of extracellular electrodes. Inhibition was slightly suppressed by adding low concentrations of the GABAA antagonists bicuculline or bicuculline methiodide to the bathing medium. Under control conditions neural activity was narrowly confined to a vertical strip of cortex. The horizontal spread of activity expanded about twofold in the presence of antagonist concentrations (less than or equal to 0.5 microM) that were expected to suppress GABAA function by no more than 10-20%. 3. At antagonist concentrations between 0.4 and 1.0 microM, evoked epileptiform activity appeared. These threshold-dose epileptiform events showed wide variations in size and duration (even at the same recording site), very variable distances of horizontal propagation, specific sites of propagation failure, reversals of propagation direction, and directional asymmetries in their probability of propagation. This contrasts with activity observed previously (Ref. 9) in high bicuculline concentrations (greater than or equal to 10 microM): large, stereotyped events that propagate reliably without decrement or reflection. 4. Intracellular recordings were obtained from pyramidal neurons in layers II/III in the presence of less than or equal to 1 microM bicuculline. Inhibitory postsynaptic potentials (IPSPs) were observed during both primary evoked responses and propagating epileptiform events and were often comparable in size and duration to those in untreated cortex. Epileptiform field potentials were always correlated with synaptic activity in single cells, but the pattern and type of PSPs varied with the form of the field potentials. Large amplitude epileptiform events coincided with an overwhelming inhibition of upper layer neurons. 5. We conclude that 1) the horizontal spread of normal cortical activity is strongly constrained by GABAA-mediated IPSPs, 2) a relatively small reduction in the efficacy of inhibition leads to a large increase in the spread of excitation, 3) initiation and propagation of synchronized epileptiform activity can occur even in the presence of robust cortical inhibition, and 4) the character of epileptiform activity is strongly affected by the influences of inhibition.

Kinins induce abnormal vascular reactivity.

Previous studies have shown that after experimental neural trauma or acute hypertension the brain produces superoxide anion radicals, and brain arterioles display endothelial lesions, dilation, and loss of normal reactivity in response to a decrease in CO2 tension. Because these abnormalities are prevented by pretreatment with free radical scavengers or inhibitors of the cyclooxygenase component of prostaglandin (PG) H synthase, arachidonic acid metabolism by PGH synthase with concomitant formation of tissue injuring oxygen radicals causes the vascular damage. The purpose of the present experiments was to determine whether kinins, which are known to stimulate arachidonate metabolism and to induce cerebral arteriolar dilation via production of superoxide anion, may be involved in initiating the cerebrovascular abnormalities produced by neural trauma in cats. The diameter and reactivity of untreated in vivo pial arterioles on one cerebral cortex was compared with the diameter and reactivity of pial arterioles on the contralateral cortex, which were pretreated topically with a competitive receptor antagonist, which is specific for kinins. Before fluid percussion neural trauma was induced, arterioles on both cerebral hemispheres constricted normally to a decrease in CO2 tension. After injury, the arterioles on the untreated cortex dilated and did not constrict in response to a decrease in arterial CO2 tension, whereas the arterioles pretreated with the kinin antagonist dilated less and displayed normal reactivity to CO2. These experiments demonstrate that a specific kinin receptor stimulates PGH synthase-dependent, free radical-mediated cerebrovascular injury. Given the ubiquitous distribution of the kallikrein-kinin system, we propose that kinins may be an important common mediator of systemic vascular injury and abnormal vascular reactivity.

Regulation of rCBF by intracortical vasoactive intestinal polypeptide-containing neurons. Immunohistochemical and hydrogen clearance study in rats.

The role of intracortical vasoactive intestinal polypeptide (VIP)-containing neurons in the regulation of cortical blood flow was investigated in rats by immunohistochemical and hydrogen clearance methods. Immunohistochemical studies revealed an intimate association between intracortical VIP-containing neurons and small blood vessels. Intracortical injection of a VIP solution (10(-5) M) produced significantly higher blood flow in the treated cortex (mean +/- standard error of the mean: 46.2 +/- 4.0 ml/100 gm/min) than in the untreated cortex (36.9 +/- 2.4 ml/100 gm/min). These data suggest that intracortical VIP-containing neurons produce dilatation of intracortical blood vessels.

5-Hydroxytryptamine innervation of vessels in the rat cerebral cortex. Immunohistochemical findings and hydrogen clearance study of rCBF.

The role of the central 5-hydroxytryptamine (5-HT) neuron system in cerebral microcirculation of the rat was examined by immunohistochemical and hydrogen clearance methods. Immunohistochemical studies demonstrated 5-HT-immunoreactive nerve fibers along intraparenchymal blood vessels (arterioles, capillaries, and venules). Ultrastructural observation revealed that 5-HT-immunoreactive terminal boutons (0.3 to 1.0 micron in diameter) made contact with the basement membrane of the capillaries. After an intracerebral injection of 5,7-dihydroxytryptamine (5,7-DHT), a neurotoxin to the 5-HT neuron system, no 5-HT-immunoreactive nerve fibers were found around the injection site with immunohistochemical techniques. With the hydrogen clearance method, the 5,7-DHT-injected cortex showed no significant change in regional cerebral blood flow (rCBF) in the presence of normocapnia, but a significant increase in rCBF with hypercapnia, compared with the untreated cortex. These facts strongly suggest that the central 5-HT neuron system has an important role in carbon dioxide reactivity of the cerebral blood vessels.

Effects of calcium on the electrical resistance and the extracellular space of cerebral cortex

Superfusion of the rabbit's cerebral cortex with an isotonic calcium chloride solution (100 m m) caused a marked drop in the electrical resistance to about one-half the original value of the surface layer of the cortex (about 0.6 mm deep). Electron micrographs prepared by freeze-substitution of calcium-superfused cortex exhibited an abundance of extracellular space (mean of 32.6% in six experiments), much more than in untreated cortex (13.7%). Asphyxiation of the cortex before freezing resulted in a transport of the extracellular material into cellular elements. Treatment of the calcium superfused cortex with a histochemical method for this ion revealed a darkly stained surface layer which may have been due to the presence of enlarged extracellular spaces containing a material of high calcium content. The enlargement of the extracellular space can fully account for the observed drop in tissue resistance caused by superfusion of the cortex with an isotonic CaCl 2 solution.

Divalent cations in cerebral impedance and cell membrane morphology

Electrical impedance of cerebral cortex was measured at 1.0 kHz with low current levels (10 −13 A per square micron of electrode surface). Changes in impedance were observed after topical application of calcium and magnesium solutions. These modifications were further evaluated by asphyxiation, and by dehydration with intravenous urea. Asphyxiation of untreated cortex caused an initial fall in resistance and raised capacitance rapidly followed by raised resistance and reduced capacitance. Repeated topical application of small amounts of calcium solutions induced a progressive rise in resistance, typically between 20 and 30% of baseline values 10–20 KΩ. This increased impedance reached a plateau after 30–50 min, and further application of calcium was without effect. By contrast with untreated cortex, asphyxiation was followed by a rapid fall in resistance to values around baseline levels. This impedance curve was thus biphasic, with an inverse relationship between the size of the calcium-induced rise and the ensuing fall during asphyxiation. On the other hand, magnesium ions in equivalent amounts exerted only minor effects on cortical impedance, and subsequent asphyxiation produced a large impedance increase, as in untreated cortex. However, magnesium ions produced effects similar to calcium in normal and asphyxiated cortex, if the cortex was first dehydrated with intravenous urea, and even though urea alone was without major effects on impedance. Prior dehydration with urea had no effect on the course of impedance changes produced by calcium. These findings are interpreted in terms of conductance of impedance measuring currents mainly through an extracellular space containing a hydrated network of macromolecules that blend with similar external coats on cell membrane, and which bear numerous cationic binding sites. Evidence is presented in electron micrographs prepared with aldehyde fixation that excess calcium ions narrow intercellular spaces and that these changes reverse with asphyxiation. No such narrowing was induced by urea. On the other hand, magnesium ions were associated with much augmented intercellular spaces. It is proposed that conductance in the extracellular space is determined by the state of its structural elements, including interdependent factors such as hydration and divalent cation binding, as well as by changes in its cross-sectional area. The preeminent role of calcium ions in altering tissue impedance is discussed in relation to membrane excitation, with competitive binding of calcium and hydrogen ions to anionic macromolecular sites. Evidence relating these results to information storage at the neuronal surface is discussed.