Anatomical localization and time course of Fos expression following soman-induced seizures

Soman (pinacolymethylphosphonofluoridate), a highly potent, irreversible inhibitor of cholinesterase, causes intense convulsions, neuropathology and, ultimately, death. There is evidence that certain brain structures are selectively vulnerable to the pathological consequences of soman-induced seizures. A working hypothesis is that central nervous system (CNS) structures with the earliest and most severe signs of neuropathology may be key sites for the initiation of the seizures. Fos, the immediate-early gene product, increases rapidly in several animal seizure models. Thus, we reasoned that the earliest brain regions to express Fos might be involved in the initiation and maintenance of soman-induced convulsions. To assess this, rats were injected with a single, convulsive dose of soman (77.7 micrograms/kg, i.m.). The animals were euthanized and processed for immunocytochemical analysis at several time points. Robust Fos expression was seen in layer II of the piriform cortex and the noradrenergic nucleus locus coeruleus within 30-45 minutes. One hour following soman injection, staining was more intense in the piriform cortex layer II and in the locus coeruleus. In addition, Fos was evident in the piriform cortex layer III, the entorhinal cortex, the endopiriform nucleus, the olfactory tubercle, the anterior olfactory nucleus and the main olfactory bulb. By 2 hours, Fos staining was present throughout the cerebral cortex, thalamus, caudate-putamen and the hippocampus. At 8 hours and beyond, Fos expression returned to control levels throughout the CNS except for the piriform cortex and the locus coeruleus which still had robust labeling. By 24 hours, neuropathology was evident throughout the rostral-caudal extent of layer II of the piriform cortex. The rapid induction of Fos in the piriform cortex and the locus coeruleus, taken together with previous anatomical, eletrophysiological and neurochemical studies, suggests that prolonged, excessive exposure to synaptically released acetylcholine and norepinephrine triggers the production of soman-induced seizures initially in the piriform cortex and subsequently in other cortical and subcortical structures.     

Antibody response and protective capacity of plasmid vaccines expressing three different herpes simplex virus glycoproteins

We have studied the morphometric changes of the neurons of the cingular area 24 and frontal area 6 of the mouse, produced by age and/or chronic alcohol intake. The parameters analyzed were nuclear area of these cortical neurons and cellular density (cell/neuropil coefficient). We detected a decrease in the number of neurons with age in practically all layers of the control animals. In the animals that chronically ingested the alcoholic solution, we also detected a decrease in the number of neurons with age, but only in layer V of the frontal cortex and in layer VI of the cingular area 24. The comparison between the control and the alcoholic group showed that alcohol intake caused an increase in the nuclear area of the neurons in layer II-III of the frontal cortex at 180 days, while in the cingular cortex the increase in nuclear area of its neurons was significative at 180 days in layer II-III and at 35 and 180 days in layers V and VI. We think that these changes are the expression of the neuronal plasticity in both cortical areas in response to the alcohol exposure.     

Sonographically detected fetal and placental abnormalities associated with trisomy 16 confined to the placenta. A case report and review of the literature

Previous studies have demonstrated that cortical spreading depression (CSD) induces neuronal tolerance to a subsequent episode of ischemia. The objective of the present investigation was to determine whether CSD alters levels of mRNA coding for putative neuroprotective proteins. Unilateral CSD was evoked in male Wistar rats by applying 2 mol/L KCl over the frontal cortex for 2 hours. After recovery for 0, 2, or 24 hours, levels of several mRNA coding for neuroprotective proteins were measured bilaterally in parietal cortex using Northern blot analysis. Levels of c-fos mRNA and brain-derived neurotrophic factor (BDNF) mRNA were markedly elevated at 0 and 2 hours, but not 24 hours after CSD. Tissue plasminogen activator (tPA) mRNA levels were also significantly increased at 0 and 2 hours, but not 24 hours after CSD. Levels of the 72-kDa heat-shock protein (hsp72) mRNA were not significantly increased by CSD, except for a small elevation (20%) at 2 hours recovery. Levels of the 73-kDa heat-shock cognate (hsc73) mRNA were slightly, but significantly, increased at 2 and 24 hours of recovery. Finally, levels of mRNA for protease nexin-1 and glutamine synthetase were not significantly altered by CSD at any time studied. The current results support the hypothesis that neuronal tolerance to ischemia after CSD may be mediated by increased expression of FOS, BDNF, or tPA, but not by increased expression of hsp72, hsc73, nexin-1, or glutamine synthetase.     

Converging inputs to the entorhinal cortex from the piriform cortex and medial septum: facilitation and current source density analysis

Converging inputs to the entorhinal cortex from the piriform cortex and medial septum: facilitation and current source density analysis. J. Neurophysiol. 78: 2602-2615, 1997. The entorhinal cortex receives sensory inputs from the piriform cortex and modulatory inputs from the medial septum. To examine short-term synaptic facilitation effects in these pathways, current source density (CSD) analysis was used first to localize the entorhinal cortex membrane currents, which generate field potentials evoked by stimulation of these afferents. Field potentials were recorded at 50-micron intervals through the medial entorhinal cortex in urethan-anesthetized rats and the one-dimensional CSD was calculated. Piriform cortex stimulation evoked a surface-negative, deep-positive field potential component in the entorhinal cortex with mean onset and peak latencies of 10.4 and 18.4 ms. The component followed brief 100-Hz stimulation, consistent with a monosynaptic response. CSD analysis linked the component to a current sink, which often began in layer I before peaking in layer II. A later, surface-positive field potential component peaked at latencies near 45 ms and was associated with a current source in layer II. Medial septal stimulation evoked positive and negative field potential components which peaked at latencies near 7 and 16 ms, respectively. A weaker and more prolonged surface-negative, deep-positive component peaked at latencies near 25 ms. The early components were generated by currents in the hippocampal formation, and the late surface-negative component was generated by currents in layers II to IV of the entorhinal cortex. Short-term facilitation effects in conscious animals were examined using electrodes chronically implanted near layer II of the entorhinal cortex. Paired-pulse stimulation of the piriform cortex at interpulse intervals of 30 and 40 ms caused the largest facilitation (248%) of responses evoked by the second pulse. Responses evoked by medial septal stimulation also were facilitated maximally (59%) by a piriform cortex conditioning pulse delivered 30-40 ms earlier. Paired pulse stimulation of the medial septum caused the largest facilitation (149%) at intervals of 70 ms, but piriform cortex evoked responses were facilitated maximally (46%) by a septal conditioning pulse 100-200 ms earlier. Frequency potentiation effects were maximal during 12- to 18-Hz stimulation of either the piriform cortex or medial septum. Occlusion tests suggested that piriform cortex and medial septal efferents activate the same neurons. The CSD analysis results show that evoked field potential methods can be used effectively in chronically prepared animals to examine synaptic responses in the converging inputs from the piriform cortex and medial septum to the entorhinal cortex. The short-term potentiation phenomena observed here suggest that low-frequency activity in these pathways during endogenous oscillatory states may enhance entorhinal cortex responsivity to olfactory inputs.     

Role of the net architecture in piriform cortex activity: analysis by a mathematical model

We present a mathematical analysis of the piriform cortex activity in rats. Experimental data were obtained by means of optical recording of fluorescent signals driven by neuronal activity. From these data, we determined the numerical value of the relaxation time for the pyramidal cell activity in layers II and III and the time latency map for bulb activation. Our model for the piriform cortex is based on pairs of excitatory and inhibitory neurons which correspond to pyramidal cells of layers II and III and to their inhibitory associated interneurons respectively; pyramidal cells are also interconnected through short and long range association fiber systems. Under such conditions, the model outputs resemble closely the experimental observations: (1) a double-bumped response to a strong and short stimulation; (2) oscillatory behavior under weak sustained stimulation conditions; (3) propagation of traveling activity waves; and (4) pacemaker activity when clusters of neurons are preferentially coupled.     

Cortical afferents of the perirhinal, postrhinal, and entorhinal cortices of the rat

We have divided the cortical regions surrounding the rat hippocampus into three cytoarchitectonically discrete cortical regions, the perirhinal, the postrhinal, and the entorhinal cortices. These regions appear to be homologous to the monkey perirhinal, parahippocampal, and entorhinal cortices, respectively. The origin of cortical afferents to these regions is well-documented in the monkey but less is known about them in the rat. The present study investigated the origins of cortical input to the rat perirhinal (areas 35 and 36) and postrhinal cortices and the lateral and medial subdivisions of the entorhinal cortex (LEA and MEA) by placing injections of retrograde tracers at several locations within each region. For each experiment, the total numbers of retrogradely labeled cells (and cell densities) were estimated for 34 cortical regions. We found that the complement of cortical inputs differs for each of the five regions. Area 35 receives its heaviest input from entorhinal, piriform, and insular areas. Area 36 receives its heaviest projections from other temporal cortical regions such as ventral temporal association cortex. Area 36 also receives substantial input from insular and entorhinal areas. Whereas area 36 receives similar magnitudes of input from cortices subserving all sensory modalities, the heaviest projections to the postrhinal cortex originate in visual associational cortex and visuospatial areas such as the posterior parietal cortex. The cortical projections to the LEA are heavier than to the MEA and differ in origin. The LEA is primarily innervated by the perirhinal, insular, piriform, and postrhinal cortices. The MEA is primarily innervated by the piriform and postrhinal cortices, but also receives minor projections from retrosplenial, posterior parietal, and visual association areas.     

Low-voltage activated T-type calcium currents are differently expressed in superficial and deep layers of guinea pig piriform cortex

A variety of voltage-dependent calcium conductances are known to control neuronal excitability by boosting peripheral synaptic potentials and by shaping neuronal firing patterns. The existence and functional significance of a differential expression of low- and high-voltage activated (LVA and HVA, respectively) calcium currents in subpopulations of neurons, acutely isolated from different layers of the guinea pig piriform cortex, were investigated with the whole cell variant of the patch-clamp technique. Calcium currents were recorded from pyramidal and multipolar neurons dissociated from layers II, III, and IV. Average membrane capacitance was larger in layer IV cells [13.1 +/- 6.2 (SD) pF] than in neurons from layers II and III (8.6 +/- 2.8 and 7.9 +/- 3.1 pF, respectively). Neurons from all layers showed HVA calcium currents with an activation voltage range positive to -40 mV. Neurons dissociated from layers III and IV showed an LVA calcium current with the biophysical properties of a T-type conductance. Such a current displayed the following characteristics: 1) showed maximal amplitude of 11-16 pA/pF at -30 mV, 2) inactivated rapidly with a time constant of approximately 22 ms at -30 mV, and 3) was completely steady-state inactivated at -60 mV. Only a subpopulation of layer II neurons (group 2 cells; circa 18%) displayed an LVA calcium current similar to that observed in deep layers. The general properties of layer II-group 2 cells were otherwise identical to those of group 1 neurons. The present study demonstrates that LVA calcium currents are differentially expressed in neurons acutely dissociated from distinct layers of the guinea pig piriform cortex.     

Increased expression of the NF2 tumor suppressor gene product, merlin, impairs cell motility, adhesionand spreading

To demonstrate regional activation in the rat cerebral cortex related to stress-evoked neuroendocrine response, Fos expression in both the cerebral cortex and hypothalamic paraventricular nucleus (PVN) was immunohistochemically examined in two experimental groups; a lipopolysaccharide (LPS) intraperitoneally injected group for inflammatory stress and a restraint group for emotional stress. The LPS injection (100 microg/100 g b.w.) and restraint (for 30 min) had similar effect on Fos-like immunoreactivity (Fos-LI) in PVN with regard to the number of immunoreactive nuclei and their distribution pattern, while the times to maximize Fos-LI were different. Numerical analysis of cortical Fos-LI in untreated rats showed a distinct region-specific pattern. Statistical analysis revealed no significant increase in Fos-LI density in any cortical regions in the LPS group, but restraint resulted in a dramatic and region-specific increase. A significant increase was detected in the prefrontal cortex (the cingulate, orbital and agranular insular cortex), the frontal area 2, the agranular retrosplenial cortex, the parietal cortex, and the medial and lateral occipital area 2. These results indicate that cortical activation relevant to specific functions may be involved in stress-specific neural circuitry.     

C-Fos, Jun D and HSP72 immunoreactivity, and neuronal injury following lithium-pilocarpine induced status epilepticus in immature and adult rats

In order to follow the maturation-related evolution of neuronal damage, cellular activation and stress response subsequent to Li-Pilo seizures in the 10- (P10), 21-day-old (P21) and adult rat, we analyzed the expression of the c-Fos protein as a marker of cellular activation, HSP72 immunoreactivity as the stress response and silver staining for the assessment of neuronal damage in 20 selected brain regions. The early wave of c-Fos measured at 2 h after the onset of seizures was present in most structures of the animals at the three ages studied and particularly strong in the cerebral cortex, hippocampus and amygdala. The late wave of c-Fos measured at 24 h after the onset of seizures and that was shown to correlate to neuronal damage was absent from the P10 rat brain, and present mainly in the cerebral cortex and hippocampus of P21 and adult rats. The expression of the stress response, assessed by the immunoreactivity of HSP72 at 24 h after the seizures was absent from the P10 rat brain and present in the entorhinal cortex, amygdala, hippocampus and thalamus of P21 and adult rats. The expression of Jun D at 24 h after the seizures was discrete and present in most brain regions at all ages. Neuronal injury assessed by silver staining at 6 h after the onset of seizures was very discrete in the brain of the P10 rat and limited to a few neurons in the piriform and entorhinal cortices. In older animals, marked neuronal degeneration occurred in the cerebral cortex, amygdala, hippocampus, lateral septum and thalamus. Thus the immediate cell activation induced by lithium-pilocarpine seizures which is present at all ages translates only into a late wave of c-Fos and the expression of HSP72 in P21 and adult animals in which there will be extensive cell damage.     

Tolerance to diazepam and changes in GABA(A) receptor subunit expression in rat neocortical areas

Long-term treatment with diazepam, a full allosteric modulator of the GABA(A) receptor, results in tolerance to its anticonvulsant effects, whereas an equipotent treatment with the partial allosteric modulator imidazenil does not produce tolerance. Use of subunit-specific antibodies linked to gold particles allowed an immunocytochemical estimation of the expression density of the alpha1, alpha2, alpha3, alpha5, gamma(2L&S) and beta(2/3) subunits of the GABA(A) receptor in the frontoparietal motor and frontoparietal somatosensory cortices of rats that received long-term treatment with vehicle, diazepam (three times daily for 14 days, doses increasing from 17.6 to 70.4 micromol/kg), or imidazenil (three times daily for 14 days, doses increasing from 2.5 to 10.0 micromol/kg). In this study, tolerance to diazepam was associated with a selective decrease (37%) in the expression of the alpha1 subunit in layers III-IV of the frontoparietal motor cortex, and a concomitant increase in the expression of the alpha5 (150%), gamma(2L&S) and beta(2/3) subunits (48%); an increase in alpha5 subunits was measured in all cortical layers. In the frontoparietal somatosensory cortex, diazepam-tolerant rats had a 221% increase in the expression of alpha5 subunits in all cortical layers, as well as a 35% increase in the expression of alpha3 subunits restricted to layers V-VI. Western blot analysis substantiated that these diazepam-induced changes reflected the expression of full subunit molecules. Rats that received equipotent treatment with imidazenil did not become tolerant to its anticonvulsant properties, and did not show significant changes in the expression of any of the GABA(A) receptor subunits studied, with the exception of a small decrease in alpha2 subunits in cortical layers V-VI of the frontoparietal somatosensory cortex. The results of this study suggest that tolerance to benzodiazepines may be associated with select changes in subunit abundance, leading to the expression of different GABA(A) receptor subtypes in specific brain areas. These changes might be mediated by a unique homeostatic mechanism regulating the expression of GABA(A) receptor subtypes that maintain specific functional features of GABAergic function in cortical cell layers.     

Stress test in the elderly. Clinical, hemodynamic, metabolic and electrocardiographic variables]

Pentylenetetrazol is a convulsive drug acting on gamma-aminobutyric acid-A (GABA[A]) gated-chloride receptors. In this study we used a subconvulsive dose (30 mg/kg) of pentylenetetrazol to induce a fully kindled state in rats. Glutamate receptors were evaluated using [3H]-[1(2-thienylcyclohexyl)]-piperidin (TCP) and [3H]kainate receptor autoradiography and [3H]muscimol autoradiography was used to study GABA(A) receptors. In fully kindled rats decreased N-methyl-D-aspartate receptor binding was found in parietal cortex, area CA2 of hippocampus and piriform cortex. Decreased kainate receptor binding was observed in all areas of the hippocampus, the medial amygdala and in the piriform cortex in the kindled rats. In contrast, GABA(A) receptor binding increased in the dentate gyrus. It is concluded that modulatory neuronal plasticity events are induced in fully pentylenetetrazol kindled rats, which appears to lead to decreased glutamatergic excitation and increased GABAergic inhibition in brain regions implicated in the development of seizure activity.     

The laminar organization of the afferent connections of the cortical column in field 17 in the cat

The laminar structure of the cortical column connections in area 17 of the cat was studied using the microiontophoretic injection of the horseradish peroxidase. Following the enzyme injection in one column at different depths below the cortical surface the identification of morphological types of labelled neurons and the estimation of their localization were performed. When enzyme has been delivered in the whole depth of the column pyramidal neurons labelled were found in upper (I/III) and lower (V/VI) layers (ratio II;I). When the depth of enzyme injection exceeded the cortex width the cells in layer IV were labelled as well. The ratio between the quantity of cells labelled in upper and lower layers was preserved. (II; I). After the enzyme injection in the upper part of the column labelled cells were found mainly in the upper layers. It is concluded that the neurons of the column of have extensive (up to 5 mm), predominantly horizontal afferent connections with the cells in upper and lower layers while the connections with neurons in layer IV are local and do not extend beyond 0.5 mm.     

Increased striatal proenkephalin mRNA subsequent to production of spreading depression in rat cerebral cortex: activation of corticostriatal pathways?

Cortical Spreading Depression (CSD) is a slowly propagating wave of depolarization and negative interstitial DC potential, that when induced in the rat brain extends across the entire homolateral hemisphere. Despite evidence that CSD does not penetrate into subcortical regions, neurochemical changes in areas anatomically connected to cortex have been reported. In this study in situ hybridization histochemistry was used to examine the levels of cholecystokinin (CCK), proenkephalin (ENK) and prodynorphin (DYN) mRNA in cortex and forebrain basal ganglia following KCl-induced CSD. Unilateral CSD was induced by topical application of 3 M KCl ( approximately 10 microliter) onto the right parietal cortex for 10 min and rats were then killed 1-6 h and 1-28 days later. CCK mRNA levels were increased (P<0.01) in the ipsilateral neocortex 3 h after CSD (13% above levels in contralateral side), reached a peak at 2 days ( approximately 70%) and were still elevated at 7 (30%) but not, 14 or 28 days later. Unilateral CSD also produced a rapid and sustained increase (P<0.05) in ENK mRNA in ipsilateral piriform cortex (from 3 h to 2 days; 70-250% above contralateral), and a delayed increase in caudate putamen and olfactory tubercle at 1 and 2 days ( approximately 25% in both regions), but levels were again equivalent to control at 7 days and beyond. In contrast, no marked changes in neocortical ENK mRNA, or DYN mRNA in both cortex and basal ganglia, were observed under these conditions. These findings demonstrate that CSD has specific, rapid and long-lasting effects on neuropeptide expression in neocortex and subcortical areas. CSD-induced changes in mesostriatal ENK mRNA are proposed to reflect synaptic activation of local neurons via cortical afferent projections.     

Filopodia and growth cones in the vertically migrating granule cells of the postnatal mouse cerebellum

The significance of cholinergic modulation for associative memory performance in the piriform cortex was examined in a study combining cellular neurophysiology in brain slices with realistic biophysical network simulations. Three different physiological effects of acetylcholine were identified at the single-cell level: suppression of neuronal adaptation, suppression of synaptic transmission in the intrinsic fibers layer, and activity-dependent increase in synaptic strength. Biophysical simulations show how these three effects are joined together to enhance learning and recall performance of the cortical network. Furthermore, our data suggest that activity-dependent synaptic decay during learning is a crucial factor in determining learning capability of the cortical network. Accordingly, it is predicted that acetylcholine should also enhance long-term depression in the piriform cortex.     

The organization and animal-vegetal asymmetry of cytokeratin filaments in stage VI Xenopus oocytes is dependent upon F-actin and microtubules

Confocal immunofluorescence microscopy with anti-cytokeratin antibodies revealed a continuous and polarized network of cytokeratin (CK) filaments in the cortex of stage VI Xenopus oocytes. In the animal cortex, CK filaments formed a dense meshwork that both was thicker and exhibited a finer mesh than the network of CK filaments previously observed in the vegetal cortex (Klymkowsky et al., 1987). CK filaments first appeared in association with germinal vesicle (GV) and mitochondrial mass (MM) of oocytes in early mid stage I, indicating that CK filaments are the last of the three cytoskeletal networks to be assembled. By late stage I, CK filaments formed complex networks surrounding the GV, surrounding and penetrating the MM, and linking these networks to a meshwork of CK filaments in the oocyte cortex. During stage III-early IV, CK filaments formed a highly interconnected, apparently unpolarized, radial array linking the perinuclear and cortical CK filament networks. Polarization of the CK filament network was observed during mid stage IV-stage V, as first the animal, then the vegetal CK filament networks adopted the organization characteristic of stage VI oocytes. Treatment of stage VI oocytes with cytochalasin B disrupted the organization of both cortical and cytoplasmic CK filaments, releasing CK filaments from the oocyte cortex and inducing formation of numerous cytoplasmic CK filament aggregates. CB also disrupted the organization of cytoplasmic microtubules (MTs) in stage VI oocytes. Disassembly of oocyte MTs with nocodazole resulted in loss of the characteristic A-V polarity of the cortical CK filament network. In contrast, disruption of cytoplasmic CK filaments by microinjection of anti-CK antibodies had no apparent effect on cytoplasmic or MT organization. We propose a model in which the organization and polarization of the cortical network of CK filaments in stage VI Xenopus oocytes are dependent upon a hierarchy of interactions with actin filaments and microtubules.