Rapid and selective detection of viruses using virus-imprinted polymer films.

We prepared a nanopatterned polymer film of polydimethylsiloxane (PDMS) via virus imprinting. The imprinted surface exhibited nanoscale cavities with the mean size of 120 ± 4 nm. These cavities demonstrated the ability to preferentially capture a target virus from an aqueous suspension of ultralow volume (5 µL) after only 1 minute of contact. Two inactivated viruses with similar shape, Influenza A (HK68) and Newcastle Disease Virus (NDV), were employed as model pathogens. The polymer film, which was first imprinted with HK68 and exposed sequentially to suspensions containing fluorescently labeled NDV and HK68, was able to preferentially bind HK68 at a capture ratio of 1 : 8.0. When we reversed the procedure and imprinted with NDV, the capture ratio was 1 : 7.6. These results were obtained within 20 minutes of static exposure. The suspensions contained viruses at concentrations close to those occurring physiologically in influenza infections. The limit of detection was approximately 8 fM. Production of virus-imprinted films can be readily scaled to large quantities and yields a disposable, simple-to-use device that allows for rapid detection of viruses.

Medial septum mediates the increase in post-ictal behaviors and hippocampal gamma waves after an electrically induced seizure.

Hippocampal EEG and behavior of freely moving rats were studied before and after a hippocampal afterdischarge (AD), with or without reversible inactivation of the medial septum (MS) by muscimol. Muscimol suppressed the normal hippocampal EEG, including theta (5-10 Hz) and gamma (30-70 Hz) waves. After a hippocampal AD, hippocampal gamma waves were decreased for about 2 min and then increased at 3-10 min, while power of EEG of <30 Hz was decreased at

Effects of a water-soluble antitumor ether phosphonoinositide, D-myo-inositol 4-(hexadecyloxy)-3(S)-methoxybutanephosphonate (C4-PI), on inositol lipid metabolism in breast epithelial cancer cell lines.

We have demonstrated previously that D-myo-inositol 4-(hexadecyloxy)-3(S)-methoxybutanephosphonate (C4-PI), an isosteric phosphonate analog of phosphatidylinositol developed to inhibit inositol lipid metabolism, was unable to inhibit phosphatidylinositol (PI) 3-kinase activity. We now report the effects of the compound on other aspects of inositol metabolism. We demonstrated that C4-PI inhibits the activity of purified recombinant PI-phospholipase C-beta (PLC-beta) at all concentrations tested; it enhanced the activity of PI-PLC-gamma and PI-PLC-delta at low concentrations (10 microM), while severely inhibiting their activities at higher concentrations. In the breast cancer cell lines MCF-7 (estrogen receptor positive) and MDA-MB-468 (estrogen receptor negative), C4-PI had no effect on the uptake of D-myo-inositol but severely inhibited its incorporation into PI. In spite of the drastic decrease in PI synthesis, C4-PI did not affect the levels of inositol incorporated into phosphatidylinositol 4,5-bisphosphate (PIP2) in the cells. In vitro assays showed that C4-PI inhibited PI synthase activity (inhibition of 35% at 50 microM) but had little effect on PI 4-kinase activity (inhibition of 13% at 150 microM). C4-PI inhibited the proliferation of MCF-7 and MDA-MB-468 cell lines with IC(50) values of 12 and 18 microM. Taken together, the results suggest that the accumulation of [3H]inositol in PIP2 in cells incubated with C4-PI may be due to the inhibition of PIP2 hydrolysis in the cells with no effect on its synthesis. The role of these C4-PI-induced effects in the mechanism of growth inhibition by C4-PI remains to be established.

A role of subicular and hippocampal afterdischarges in initiation of locomotor activity in rats.

The possible role of a hippocampal afterdischarge (AD) episode in eliciting locomotor movements was evaluated in freely moving rats. Electrical stimulation of either the ventral subiculum (VSB) or the hippocampal CA1 region evoked an AD of 6-50 s in duration, which was followed by an increase in locomotor activity. Similar results were also observed after unilateral injection of N-methyl-d-aspartic acid (NMDA, 0.25 microg or 1 microg), a glutamate receptor agonist, into the VSB. Locomotor activity was not observed when either electrical or chemical stimulation of the VSB, or electrical stimulation of the CA1 region did not elicit an AD. In addition, the duration of the AD was positively correlated with the number of locomotor movements induced by stimulation of VSB or CA1 region. It is suggested that the hippocampal/subicular AD may be a necessary condition to induce locomotor activity by either chemical or electrical stimulation of the hippocampus in rats.

A convenient synthesis of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and L-myo-inositol 1,4,5-trisphosphate (Ins(3,5,6)P3).

An efficient synthesis of an optically active inositol derivative that is a precursor to D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3, (-)) is described. Crystallization of the diastereomers of (+/-)-1-O-[(+)-menthoxycarbonyl]-6-O-benzyl-2,3:4,5-di-O-isopropyl idene-myo- inositol diastereomers from methanol gives only one diastereomer. Alkaline hydrolysis gives the useful inositol derivative (-)-6-O-benzyl-2,3:4,5-di-O-isopropylidene-myo-inositol. Likewise, crystallization of the diastereomers of (+/-)-3-O-[(-)-menthoxycarbonyl]-4-O-benzyl-1,2:5,6-di-O-isopropyl idene-myo- inositol from methanol gave a pure compound which could be hydrolyzed to give (+)-4-O-benzyl-1,2:5,6-di-O-isopropylidene-myo-inositol, a precursor to D-myo-inositol 3,5,6-trisphosphate (Ins(3,5,6)P3,(+)). The ease with which these enantiomerically pure inositol derivatives were isolated may facilitate the synthesis of more complex inositol phosphate derivatives such as D-myo-inositol 1,3,4,5-tetrakisphosphate.

Involvement of the nucleus accumbens-ventral pallidal pathway in postictal behavior induced by a hippocampal afterdischarge in rats.

The hypothesis that postictal motor behaviors induced by a hippocampal afterdischarge (AD) are mediated by a pathway through the nucleus accumbens (NAC) and ventral pallidum (VP) was evaluated in freely moving rats. Tetanic stimulation of the hippocampal CA1 evoked an AD of 15-30 s and an increase in number of wet-dog shakes, face washes, rearings and locomotor activity. Bilateral injection of haloperidol (5 micrograms/side) or the selective dopamine D2 receptor antagonist, (+/-)-sulpiride (200 ng/side) before the hippocampal AD, into the NAC selectively reduced rearings and locomotor activity, but not the number of wet-dog shakes and face washes. Injection of R(+)-SCH-23390 (1 microgram/side), a D1 receptor antagonist, or rimcazole (0.4 mg/side), a sigma opioid receptor antagonist, into the NAC did not significantly alter postictal behaviors. Bilateral injection of muscimol (1 ng/side), a gamma-aminobutyric acid (GABAA) receptor agonist, into the VP before the AD significantly blocked all postictal behaviors. It is concluded that postictal locomotor activity induced by a hippocampal AD is mediated by activation of dopamine D2 receptors in the NAC and a pathway through the VP.

Evaluation of the hypothesis that hippocampal interictal spikes are caused by long-term potentiation.

Hippocampal spontaneous interictal spikes (SISs) occur in the EEG after repeated afterdischarges (ADs) induced by high-frequency (200 Hz) stimulation trains. Because SISs resemble population excitatory postsynaptic potentials (EPSPs), and SISs persist for several days like some types of hippocampal long-term potentiation (LTP), LTP has been suggested as a mechanism for SISs. We specifically examined the hypothesis that SISs are caused by basal-dendritic LTP in CA1. Rats were chronically implanted with bilateral electrodes in the hippocampal CA1 region. In the first experiment, 10-18 patterned primed burst (PB) stimulations were delivered hourly for 2-3 days to activate the commissural basal-dendritic EPSP in CA1. Robust LTP of the basal-dendritic CA1 synapse was detected, typically saturating at 100% enhancement after five stimulations. However, few SISs were detected if ADs were not elicited. In the second experiment, repeated commissurally evoked ADs induced a high rate of SISs, together with LTP of the basal-dendritic and apical-dendritic EPSP in CA1, but the SIS rate was not necessarily related to the level of LTP. In the third experiment, an intraventricular dose (20 micrograms) of an N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (APV) was used to block the basal-dendritic LTP in CA1. The increase in SISs induced by a single AD was not blocked, however, suggesting that NMDA receptors were not critical in generation of SISs. In the fourth experiment, PBs (that induced LTP but no ADs) were able to increase the rate of SISs marginally when SISs were already present. In all, the experiments suggest that LTP at the basal dendrites of CA1 is not critical in generation of hippocampal SISs, although an increase in LTP may increase the rate of SISs marginally when SISs are already present.

A movement-associated fast rolandic rhythm.

A stereotyped 32 Hz rolandic rhythm maximal over the mesial cortex and occurring only with voluntary movement is described in a patient with tonic postural seizures. This abnormal but nonictal rhythm, which is probably generated subcortically, is expressed synchronously at the level of the cortex and peripherally in the EMG.

Intrinsic membrane potential oscillations in hippocampal neurons in vitro.

Membrane potential oscillations (MPOs) of 2-10 Hz and up to 6 mV were found in almost all stable hippocampal CA1 and CA3 neurons in the in vitro slice preparation. MPOs were prominent for pyramidal cells but less pronounced in putative interneurons. MPOs were activated at threshold depolarizations that evoked a spike and the frequency of the MPOs increased with the level of depolarization. MPOs were distinct from and seemed to regulate spiking, with a spike often riding near the top of a depolarizing MPO wave. Analysis of the periodicity of the oscillations indicate that the period of MPOs did not depend on the afterhyperpolarization (AHP) following a single spike. MPOs persisted in low (0-0.1 mM) Ca2+ medium, with or without Cd2+ (0.2 mM), when synaptic transmission was blocked. Choline-substituted low-Na+ (0-26 mM) medium, 3 microM tetrodotoxin (TTX) or intracellular injection of QX-314 reduced or abolished the fast Na(+)-spike and reduced inward anomalous rectification. About 40% of CA1 neurons had no MPOs after Na+ currents were blocked, suggesting that these MPOs were Na(+)-dependent. In about 60% of the cells, a large depolarization activated Ca(2+)-dependent MPOs and slow spikes. MPOs were not critically affected by extracellular Ba2+ or Cs2+, or by 0.2 mM 4-aminopyridine, with or without 2 mM tetraethylammonium (TEA). However, in 5-10 mM TEA medium, MPOs were mostly replaced by 0.2-3 Hz spontaneous bursts of wide-duration spikes followed by large AHPs. Low Ca2+, Cd2+ medium greatly reduced the spike width but not the spike-bursts. In conclusion, each cycle of an MPO in normal medium probably consists of a depolarization phase mediated by Na+ currents, possibly mixed with Ca2+ currents activated at a higher depolarization. The repolarization/hyperpolarization phase may be mediated by Na+/Ca2+ current inactivation and partly by TEA-sensitive, possibly the delayed rectifier, K+ currents. The presence of prominent intrinsic, low-threshold MPOs in all hippocampal pyramidal neurons suggests that MPOs may play an important role in information processing in the hippocampus.

Spontaneous hippocampal interictal spikes following local kindling: time-course of change and relation to behavioral seizures.

Spontaneous interictal spikes (SISs) were recorded in the hippocampus in freely behaving rats following hippocampal stimulations that resulted in afterdischarges (ADs). Hippocampal SISs were detected after an average of 5 (range 2-10) daily ADs. The rate of SISs typically increased minutes after a tetanus, and then decayed with time constants of approximately 70 min and 1.5 days. Seizure onset in the kindling paradigm was not related to a consistent change in SIS rate. Following the interruption of daily kindling, SIS rate invariably decreased to near zero by 4-8 days while seizure susceptibility, as tested by the ability to evoke generalized convulsions, remained unchanged. Despite having a low or zero SIS rate the hippocampus seemed to retain an excitability after kindling interruption, as demonstrated by the observation that an average of 1.7 rekindling stimulations resulted in a high SIS rate. In conclusion, changes in hippocampal SISs were closely time-locked to an AD, and not to evoked behavioral seizures. Hippocampal SISs probably reflect an excitability change that is more local than that necessary for evoking behavioral convulsions. The persistence of SISs in terms of hours and days suggests the involvement of long-term potentiation.

The role of serotonin in the control of cerebral activity: studies with intracerebral 5,7-dihydroxytryptamine.

Intact rats treated with centrally acting antimuscarinic (atropinic) drugs display large amplitude irregular slow waves in both the neocortex and hippocampus during behavioral immobility and some stereotyped automatic behaviors (Type 2 behavior). However, rhythmical slow activity in the hippocampus and low voltage fast activity in the neocortex occur in close correlation with spontaneous changes in posture, head movement, walking, rearing, swimming or struggling when held (Type 1 behavior). It has previously been proposed that these waveforms, jointly referred to as atropine-resistant cerebral activation (ARCA) are dependent on ascending serotonergic projections. As a further test of this hypothesis, we have studied rats in which forebrain levels of serotonin and 5-hydroxyindoleacetic acid were reduced to 3-10% of control levels as a result of multiple intrabrainstem injections of 5,7-dihydroxytryptamine. This treatment strongly reduced or abolished ARCA in most cases but did not reduce atropine-sensitive cerebral activation which appears to be dependent on ascending cholinergic projections from the basal forebrain to the cerebral cortex. Therefore, ARCA appears to be dependent on ascending serotonergic inputs to the forebrain.

Hippocampal interictal spikes induced by kindling: relations to behavior and EEG.

Hippocampal spontaneous interictal spikes (SISs) were recorded during the course of daily tetanization (kindling) of afferent fibers to the hippocampal CA1 region. SISs were detected after 3-10 tetanizations. A clear variation of SIS rate with behavior was observed. SIS rate was high during slow-wave sleep (SWS), waking immobility, face-washing and chewing and low during rapid-eye-movement sleep (REMS), walking and rearing. Scopolamine hydrochloride (2.5-5 mg/kg i.p.) increased the SIS rate during walking. Despite the negative correlation of SIS occurrence with the theta rhythm in normal rats, abolishing the theta rhythm by medial septal lesions did not affect the suppression of SISs during REMS as compared to SWS. When interictal or postictal spikes were seen together with the theta rhythm, the spikes tended to occur at a phase of about 240 degrees after the positive peak of the alvear surface rhythm.

APV, an N-methyl-D-aspartate receptor antagonist, blocks the hippocampal theta rhythm in behaving rats.

2-Aminophosphonovaleric acid (APV), an N-methyl-D-aspartate (NMDA) receptor antagonist, was infused into the lateral ventricles of behaving rats. A 10 or 20 microgram dose of APV attenuated the hippocampal theta rhythm and the theta phase-shift at the apical dendrites of hippocampal CA1 region. A selective suppression of the atropine-sensitive theta rhythm was suggested.

Hippocampal electrical activity in the diabetes insipidus (Brattleboro) rat.

Theta rhythm was recorded from the hippocampus in normal male and female rats, and from female rats with homozygous alleles (HODI) and heterozygous alleles of diabetes insipidus (HEDI). Second-by-second spectral analysis of the complete period of rapid eye-movement sleep indicated that HODI and HEDI rats had the same theta frequency range as normals, but the mean theta frequency (6.4 cycles c/s) was lower than normal (6.8 c/s), mainly in having a smaller proportion of frequencies greater than or equal to 7.8 c/s. Pharmacological studies in the waking rat demonstrated a theta rhythm in the HODI and normal rats after atropine or after urethane and eserine, indicating the presence of both atropine-sensitive and atropine-resistant pathways. However, after eserine, a huge increase in hippocampal fast waves (30 to 55 c/s) accompanying struggling (as compared with immobility) was found in the HODI rat, which was double that in the normal rat. An enhanced cholinergic input or response at the septal or hippocampal level may account for the large fast wave as well as the lower mean theta frequency in the HODI rat.

Hippocampal electrical activity following local tetanization. I. Afterdischarges.

Following a short (1-10 s) train of repetitive stimulation delivered to the hippocampal CA1 region, the following sequelae of afterdischarges (ADs) was seen: (1) a silent period of 2-4 s, (2) a large primary (1 degree) AD usually alvear-surface negative and deep positive, (3) a period of suppressed hippocampal EEG, (4) a secondary (2 degrees) hippocampal AD, and after 3-6 min, (5) 15-25 min of enhanced (up to 10 times normal) fast (30-70 Hz) waves. The 2 degrees hippocampal AD was preceded by or simultaneous with large AD at the amygdaloid electrodes. Electrolytic lesions (n = 7) or large heat lesions of the amygdala (n = 5) or electrolytic lesions of the medial septum (n = 10) were not successful in suppressing the 2 degrees hippocampal AD. However, 4 rats with radiofrequency lesion and 3 rats with bilateral aspiration lesion of the entorhinal cortex had diminished or no 2 degrees hippocampal AD. The fast waves after tetanization were reversed 180 degrees across surface and deep CA1 electrodes. The fast wave increase was blocked by atropine sulfate (25-50 mg/kg i.p.), scopolamine hydrochloride (5 mg/kg i.p.) and medial septal lesions. It was concluded that the 2 degrees hippocampal AD may depend on a reverberation of neural circuitry involving the entorhinal cortex. The 2 degrees AD recorded from amygdala electrodes may partly reflect spreading of activities from the entorhinal cortex. On the other hand, the increase in fast waves after tetanization requires an intact septohippocampal, muscarinic cholinergic input, and may depend on an enhanced cholinergic input or an increased response.

Transcallosal evoked potentials in relation to behavior in the rat: effects of atropine, p-chlorophenylalanine, reserpine, scopolamine and trifluoperazine.

Single pulse electrical stimulation of the sensorimotor cortex in waking rats produced an evoked response in the contralateral sensorimotor cortex. The slow wave response consisted of: (1) an early component that was negative at the pial surface and in layer V, and was associated with multiunit discharge; and (2) a late component that was mainly negative at the surface, positive in layer V, and was associated with multiunit suppression. Previous research suggests that the early component represents summed excitatory postsynaptic potentials; the late component summed inhibitory postsynaptic potentials. Both components could be elicited by direct stimulation of the corpus callosum and both were abolished by midline callosal section. The amplitude and duration of the late component varied with concurrent motor activity in a striking manner. It was large during waking immobility and also during face-washing, licking the paws, chewing food and drinking water, but was much reduced or absent during head movements, walking and changes in posture. Only minor changes were associated with the transition from waking immobility to slow wave sleep. A series of pharmacological experiments indicated that the behavior-related variation in the late component of the transcallosal evoked response was dependent on both cholinergic and serotonergic transmission.

Electrical activity of the cingulate cortex. I. Generating mechanisms and relations to behavior.

Spontaneous slow waves (EEG) and multiple unit activity (MUA) were recorded in the posterior cingulate cortex (area 29) and the dorsal hippocampus of the freely moving rat by means of chronically implanted electrodes. Three different wave patterns were discerned in the cingulate EEG. Irregular slow waves occurred during grooming, drinking, eating (Type II behavior) and slow-wave sleep (SWS). The irregular waves also contained sharp transients of about 20 ms duration called EEG-spikes. EEG-spikes reversed their polarity within the cingulate cortex and correlated with an increase in cingulate MUA. They were probably generated by deep (layer IV to VI) neurons in the cingulate cortex. Theta rhythm of 6-10 Hz accompanied walking, rearing, postural shifts, head movements (Type I behavior) and rapid-eye-movement sleep (REMS). MUA of low-amplitude units was phase-locked to the local theta waves, suggesting local generation of the slow waves. However, volume-conduction from the hippocampus would likely contribute to the cingulate theta since no reversal of the theta waves was found in the cingulate cortex. Fast waves of greater than 30 Hz were generally larger during Type I than during Type II behavior. Cellular generators for fast waves are not known. High-amplitude (greater than 100 microV) MUA only appeared during Type II behavior, and in particular during SWS. During REMS, these units were silent. Stimulation of the contralateral homotopic cingulate cortex gave antidromic and synaptic components in the average evoked potential (AEP). The long latency waves of the AEP varied with behaviors and appeared oscillatory (25-40 Hz) during Type I but not during Type II behavior. In summary, the cingulate cortex has a rich gamut of spontaneous and evoked electrical activities which bears some resemblance to that of the hippocampus.

Electrical activity of the cingulate cortex. II. Cholinergic modulation.

The role of the cholinergic innervation in the modulation of cingulate electrical activity was studied by means of pharmacological manipulations and brain lesions. In the normal rat, an irregular slow activity (ISA) accompanied with EEG-spikes was recorded in the cingulate cortex during immobility as compared to walking. Atropine sulfate, but not atropine methyl nitrate, increased ISA and the frequency of cingulate EEG-spikes. Pilocarpine suppressed ISA and EEG-spikes during immobility, and induced a slow (4-7 Hz) theta rhythm. Unilateral or bilateral lesions of the substantia innominata and ventral globus pallidus area using kainic acid did not significantly change the cingulate EEG or its relation to behavior. Large electrolytic lesions of the medial septal nuclei and vertical limbs of the diagonal band generally decreased or abolished all theta activity in the cingulate cortex and the hippocampus. However, in 5 rats the cingulate theta rhythm increased while the hippocampal theta disappeared after a medial septal lesion. The large, postlesion cingulate theta, accompanied by sharp EEG-spikes during its negative phase, is an unequivocal demonstration of the existence of a theta rhythm in the cingulate cortex, independent of the hippocampal rhythm. Cholinergic afferents from the medial septum and diagonal band nuclei are inferred to be responsible for the behavioral suppression of cingulate EEG-spikes and ISA, and partially for the generation of a local cingulate theta rhythm. However, an atropine-resistant pathway and a theta-suppressing pathway, possibly coming from the medial septum or the hippocampus, may also be important in cingulate theta generation.

Topographical projection of cholinergic neurons in the basal forebrain to the cingulate cortex in the rat.

The cholinergic innervation of the rat's posterior cingulate cortex (Brodmann's area 29) was studied using acetylcholinesterase (AChE) histochemistry. Electrolytic lesion of the ipsilateral medial septum and diagonal band region (MS-DB) reduced the diffuse AChE staining in layers I, II, III and V of the cingulate cortex. Kainic acid lesion of the ipsilateral globus pallidus and substantia innominata area (GP-SI) abolished the dense band of AChE stain in layer IV, with small reductions of AChE stain in other layers. The results indicate that the medial cholinergic pathway from MS-DB terminates diffusely in layers I, II, III and V while the lateral cholinergic pathway from the GP-SI predominantly ends in layer IV of the posterior cingulate cortex.