Fluorescence image and microbiological analysis of biofilm retained around healthy and inflamed orthodontic miniscrews.

INTRODUCTION: Peri-miniscrew inflammation is one of the causes of orthodontic miniscrew failure. OBJECTIVE: The aim of this study was to correlate and quantify throughout autofluorescence images, PCR and microbiologic count of biofilm retained around orthodontic miniscrew and the presence of Porphyromonas gingivalis. MATERIALS AND METHODS: Forty miniscrews used for orthodontic treatment were evaluated during orthodontic treatment, collected from patients and divided into two groups: healthy and inflamed miniscrews. To be considered inflamed, the samples should present: loss of stability checked by periotest®, clinical presence of mucositis, red aspect of the gum or bleeding around the miniscrew. Immediately after removal of the miniscrews, they were photographed using a macro 100 lens and a Pentax camera coupled to a fluorescent equipment - Qscan (AioBio - Korea) with 405 nm excitation wavelength and a blue band filter. A microbiologic sample was collected with a sterile microbrush scrubbed around the miniscrew tread. Fluorescent images were analyzed with ImageJ software to quantify fluorescent intensity and fluorescent area and microbiological samples were submitted to CFU count for total contamination and q-PCR assay to quantify Porphyromonas gingivalis. RESULTS: The results showed a good correlation between CFU count and fluorescent intensity and PCR/fluorescent area. The healthy miniscrews presented less fluorescent intensity and lower CFU count when compared to inflamed miniscrews. q-PCR analysis showed a higher number of P. gingivalis contamination around inflamed miniscrews. CONCLUSION: Quantification of biofilm retained by miniscrew by images of autofluorescence is a simple and reliable method with great potential for clinical use to monitory inflammation around miniscrew and risk of loss.

Efficient signal processing of multineuronal activities for neural interface and prosthesis.

OBJECTIVES: Multineuronal spike trains must be efficiently decoded in order to utilize them for controlling artificial limbs and organs. Here we evaluated the efficiency of pooling (averaging) and combining (vectorizing) activities of multiple neurons for decoding neuronal information. METHODS: Multineuronal activities in the monkey inferior temporal (IT) cortex were obtained by classifying spikes of constituent neurons from multichannel data recorded with a multisite microelectrode. We compared pooling and combining procedures for the amount of visual information transferred by neurons, and for the success rate of stimulus estimation based on neuronal activities in each trial. RESULTS: Both pooling and combining activities of multiple neurons increased the amount of information and the success rate with the number of neurons. However, the degree of improvement obtained by increasing the number of neurons was higher when combining activities as opposed to pooling them. CONCLUSION: Combining the activities of multiple neurons is more efficient than pooling them for obtaining a precise interpretation of neuronal signals.

Effect of long-term potentiation induction on gamma-band electroencephalograms in prefrontal cortex following stimulation of rat hippocampus in vivo.

We examined whether long-term potentiation (LTP) affects cortical gamma-band electroencephalograms (EEG) in the hippocampo-prefrontal cortex (PFC) pathway of anesthetized rats. The LTP induction increased the evoked PFC gamma-band EEG power (40-100 Hz) to 120-135% at 500-700 ms after test stimulation. A simple increment of stimulus intensity, instead of LTP induction, did not reveal this evoked increase. Neither LTP induction nor the intensity increment changed significantly the magnitude of an evoked decrease at around 100 ms or the spontaneous prestimulation gamma-band power. These results indicate that LTP in PFC specifically increases the evoked gamma-band EEG power, which may reflect a phasic mode of plastic neurotransmission through the hippocampo-PFC pathway in vivo.

Induction of stable long-term depression in vivo in the hippocampal-prefrontal cortex pathway.

We studied excitatory field potentials in the medial prefrontal cortex (mPFC, prelimbic area) to electrostimulation of the ventral hippocampus (CA1/subicular region) in the anaesthetized rat. Nine hundred stimulus trains (5 pulses at 250 Hz) applied at 1 Hz to the ventral hippocampus significantly and persistently depressed the amplitude and maximal slope ( approximately 55% for each index) of the prelimbic field potentials, but did not change the latency of the maximal slope or peak negativity. Twelve stimulus trains (50 pulses at 250 Hz) applied subsequently at 0.1 Hz restored the depression back to control level, and this reversible depression was maintained for at least 13 h. Cumulative depressive effects on the prelimbic field potential amplitude and maximal slope were observed upon addition of stimulus trains in the hippocampus. An important implication of the results is that the direct pathway from the hippocampus to the mPFC in the rat retains long-term depression (LTD) as a neuroplastic form in vivo. This form could cooperate with long-term potentiation (LTP) and such a bi-directional synaptic plasticity in the prefrontal cortex contributes to how cortical neural networks store information.

Multineuronal spike classification based on multisite electrode recording, whole-waveform analysis, and hierarchical clustering.

We proposed here a method of multineuronal spike classification based on multisite electrode recording, whole-waveform analysis, and hierarchical clustering for studying correlated activities of adjacent neurons in nervous systems. Multineuronal spikes were recorded with a multisite electrode placed in the hippocampal pyramidal cell layer of anesthetized rats. If the impedance of each electrode site is relatively low and the distance between electrode sites is sufficiently small, a spike generated by a neuron is simultaneously recorded at multielectrode sites with different amplitudes. The covariance between the spike waveform at each electrode site and a template was calculated as a damping factor due to the volume conduction of the spike from the neuron to the electrode site. Calculated damping factors were vectorized and analyzed by hierarchical clustering using a multidimensional statistical test. Since a cluster of damping vectors was shown to correspond to an antidromically identified neuron, spikes of different neurons are classified by referring to the distributions of damping vectors. Errors in damping vector calculation due to partially overlapping spikes were minimized by successively subtracting preceding spikes from raw data. Clustering errors due to complex spike bursts (i.e., spikes with variable amplitudes) were avoided by detecting such bursts and then using only the first spike of a burst for clustering. These special procedures produced better cluster separation than conventional methods, and enabled multiple neuronal spikes to be classified automatically. Waveforms of classified spikes were well superimposed. We concluded that this method is particularly useful for separating the activities of adjacent neurons that fire partially overlapping spikes and/or complex spike bursts.

Immunohistochemical analysis on the role of adenosine A1 receptors in epilepsy.

Adenosine has an anticonvulsant effect in various models of epilepsy. This effect appears to be mediated through the activation of adenosine A1 receptors (A1Rs). We immunohistochemically investigated the changes of A1Rs expression in kainate-treated and hippocampus-kindled rats as chronic models of epilepsy. In the normal hippocampus, a predominant expression of A1Rs was detected in the CA2/CA3a field. The A1Rs immunoreactivity in this field began to decline drastically approximately 4 weeks after kainate treatment and remained minimal 8 weeks after treatment. In the hippocampus-kindled animals, A1Rs expression was minimal in the stimulated side but remained high in the nonstimulated side. The reduced expression of A1Rs in the CA2/CA3a field may be related to chronic epileptogenesis.

Lasting effect of NO on glutamate release in rat striatum revealed by continuous brain dialysis.

The effect of nitric oxide (NO) on glutamate release in the brain of freely moving rats was investigated using a new, high time-resolution microdialysis system. Coperfusion with veratridine (VER) and NO donors increased glutamate release above than that obtained with VER alone. When steady-state levels were regained after co-perfusion, perfusion of VER alone further potentiated glutamate release. The effect depended on the initial level of VER-induced glutamate release, and was maximum for intermediate glutamate levels. These results suggest that NO influences the glutamate release system by affecting the level of neural activity and that its effect lasts and increases when steady-state levels are regained in rat striatum.

Effect of retinyl acetate on the assembly of the fibronectin extracellular matrix and the processing of the fibronectin receptor beta subunit of confluent C3H/10T1/2 mouse embryo fibroblasts.

The mouse embryo fibroblast cell line, C3H/10T1/2, synthesized and deposited a large amount of fibronectin especially in the pericellular matrix. Confluent cultures of these cells cultured in the presence of 0.3 micrograms/ml of retinyl acetate released cell surface fibronectin and the extracellular matrix fibronectin fibrils were disorganized. The immunoblot analysis demonstrated that the number of the fibronectin receptor was decreased in the prolonged culturing of retinyl acetate-treated cells. Immunoprecipitation of 35S-methionine pulse-chase labeled cell extracts by antifibronectin receptor antibody indicated that about one-half of the pre-beta subunit was processed and converted to the mature form in control cells, and only about one-fourth of the pre-beta subunit was processed in the retinyl acetate-treated confluent cells. 1-deoxymannojirimycin (MNJ), which is an inhibitor of oligosaccharide processing, induced disorganization of the extracellular matrix fibronectin assembly similar to that observed with retinyl acetate. The results of this study suggest that a mechanism of action of retinyl acetate is inhibition of the glycosylation during processing of the fibronectin receptor, a step necessary for fibronectin binding and for assembly of the extracellular matrix.

Role of pontomedullary reticular formation neurons in horizontal head movements: an ibotenic acid lesion study in the cat.

Single-cell recording, electrolytic lesion and electrical stimulation studies have indicated that the pontomedullary reticular formation (PMRF) plays a role in head movement (HM) control. However, recent studies utilizing excitotoxin lesions of the PMRF have reported no effect on HM. In the present study, we have examined the acute and chronic motor effects of injecting ibotenic acid (IBO) into the nucleus reticularis pontis oralis, nucleus reticularis pontis caudalis and rostral medullary nucleus gigantocellularis of the feline PMRF. IBO injections in all of these regions induced tonic flexion of the head toward the ipsilateral side. This effect lasted 4-16 h. It was followed by a second phase in which head flexion and whole body circling were directed toward the contralateral side. Although this forced contralateral head turning disappeared within two days, the tendency to turn contralaterally and the impaired ability to make rapid ipsilateral HMs were present throughout survival periods lasting more than 4 months. Unilateral IBO PMRF lesions reduced the amplitude of vestibular induced quick phase (anti-compensatory) HMs toward the ipsilateral side and resulted in abnormally large and persistent slow compensatory HMs toward the contralateral side. Following IBO injections, the threshold intensity for HMs evoked by electrical stimulation at the injection site was elevated, and the amplitude and velocity of evoked HMs reduced. Histological data indicated that the reticular area involved in HM control was relatively large and probably extended beyond the PMRF region examined here. However, lesions including the nucleus reticularis pontis caudalis (NRPC) produced more severe and persistent HM deficits than those including the nucleus reticularis gigantocellularis. These data together with available anatomical and electrophysiological evidence indicate that PMRF neurons play a critical role in the generation of fast horizontal HMs toward the ipsilateral side.

Anatomical distribution and response patterns of reticular neurons active in relation to acoustic startle.

A population of reticulospinal neurons with short latency response to startle-inducing stimuli was identified in the nucleus reticularis pontis caudalis (NRPC) and nucleus gigantocellularis (NRGC) of the medial pontomedullary reticular formation. The threshold and magnitude of response to auditory stimuli was correlated in these cells and in the muscles mediating startle. Startle-related neurons were significantly more likely to have high conduction velocity spinal projections than adjacent cells not related to startle. Startle-related cells were not 'dedicated' to startle, but were active in relation to spontaneous movements. Both the unit response of the startle-related cells and the startle response recorded in muscles were suppressed by the prior presentation of a weak prepulse. Thus, prepulse inhibition of startle occurs at, or prior to, the medial pontomedullary reticular formation. We conclude that these reticulospinal cells convey the output of the brainstem system modulating and triggering startle.

Spontaneous EEG spikes in the normal hippocampus. IV. Effects of medial septum and entorhinal cortex lesions.

Spontaneous EEG spikes (SPKs) were recorded from the CA1 region of the dorsal hippocampus in normal rats during behavioral states not accompanied by rhythmical slow activity (RSA) such as awake immobility and slow wave sleep. The present experiment was designed to examine the effects of large electrolytic lesions of the 2 major hippocampo-petal systems, medial septum (MS) and entorhinal cortex (EC), on spontaneous SPK activity. MS lesions, while completely abolishing RSA, did not eliminate SPKs or change their behavioral correlates. SPKs were still suppressed during behaviors (walking, head movement, etc.) normally associated with RSA. However, SPK frequency was approximately halved after MS lesions. Total bilateral EC lesions did not, in general, change SPKs or RSA unless abnormal hippocampal activities persisted over the first post-lesion week during which most of the recordings were made. Laminar profiles of SPKs in both MS- and EC-lesioned rats showed a normal pattern; small positivity in stratum oriens, large negativity in stratum radiatum and polarity reversal around stratum pyramidale. These results suggest that (1) neither medial septal input nor entorhinal cortical input to the hippocampus is necessary for the generation of SPKs, and (2) SPK suppression still occurs in the absence of these inputs during behaviors normally associated with RSA. The possibility remains, however, that in intact animals these inputs do influence SPK activity in a behavior-dependent manner.

Spontaneous EEG spikes in the normal hippocampus. V. Effects of ether, urethane, pentobarbital, atropine, diazepam and bicuculline.

Spontaneous EEG spikes (SPKs) were recorded from the CA1 region of the dorsal hippocampus in normal rats during behavioral states not accompanied by rhythmical slow activity (RSA) such as awake immobility and slow wave sleep. In the present study we examined the effects of various systemically administered drugs on hippocampal SPK activity. Three general anesthetics (ether, urethane and pentobarbital) all reduced SPK activity. At anesthetic doses both ether and pentobarbital completely abolished SPKs. This SPK abolition during anesthesia seemed qualitatively different from RSA-related SPK suppression in undrugged animals in that unlike the latter case RSA generation was not the cause of SPK abolition in the former case. Atropine (50-100 mg/kg, i.p.) did not change greatly SPK activity or its behavioral correlation. Diazepam (5-10 mg/kg, i.p.) increased amplitude and decreased frequency of hippocampal fast EEG activity, and reduced SPK activity. These effects may be based on the known action of diazepam to enhance GABA-mediated postsynaptic inhibition. At subepileptic doses (1-6 mg/kg, i.p.) the GABA antagonist bicuculline enhanced dramatically SPK-related activities. Particularly, SPK-concurrent population burst discharges in the pyramidal cell layer were greatly increased in amplitude and complexity after bicuculline. These results suggest that the SPK generation mechanism in the hippocampus is sensitive to the level of GABA-mediated inhibition.

Spontaneous EEG spikes in the normal hippocampus. II. Relations to synchronous burst discharges.

Spontaneous EEG spikes (SPKs) were recorded from the CA1 region of the dorsal hippocampus in normal rats during awake immobility and slow wave sleep. These SPKs were accompanied by synchronous burst discharges in the pyramidal cell layer. These discharges are called 'population bursts (PBs)' in that they seem to require a population of synchronously bursting neurons. PBs were classified into 2 forms on the basis of their morphologies. One form (mixed burst or MB) consisted of a mixture or superimposition of action potential bursts from a relatively small number of neurons. The other form (ripple) was a series of 3-13 (typically 5-8) high frequency (125-250 Hz) waves, usually waxing and waning. Unit action potentials were superimposed mainly on negative portions of these high frequency waves. The ripple was considered to represent summed activity of highly synchronized complex spike bursts from a relatively large number of pyramidal cells. The similarity in wave structure between these non-pathological ripples and multipeaked, epileptiform (interictal) field potentials recorded from the penicillin-treated hippocampus suggests that they may share some common underlying mechanisms.

Spontaneous EEG spikes in the normal hippocampus. III. Relations to evoked potentials.

Spontaneous EEG spikes (SPKs) were recorded from the CA1 region of the dorsal hippocampus in normal rats during behavioral states not accompanied by rhythmical slow activity (RSA). SPKs were positive in stratum oriens, negative in stratum radiatum and accompanied by population bursts (PBs) in stratum pyramidale. In order to examine the origin of SPKs and PBs single pulse or brief high frequency electrical stimuli were applied to the Schaffer collateral/commissural pathway. Evoked potentials were recorded and compared with spontaneous SPKs and PBs. The results indicate the following: (1) the laminar amplitude profile of spontaneous SPKs was similar to that of population EPSPs evoked by stimulation of the Schaffer collateral/commissural pathway; (2) the population EPSP most similar to the spontaneous SPK was evoked by a brief (20-60 msec) train of high frequency (125-500 Hz) pulses; (3) the same pattern of stimulation was also found to be most efficient in evoking a series of multiple population spikes resembling a type of spontaneous PB (ripple). These observations suggest that SPKs and PBs in CA1 represent population EPSPs and multiple population spikes, respectively and that these CA1 events are triggered by brief, high frequency burst discharges of CA3 pyramidal cells via the Schaffer collateral and commissural pathway.

Spontaneous EEG spikes in the normal hippocampus. I. Behavioral correlates, laminar profiles and bilateral synchrony.

Spontaneous EEG and unit activities were recorded from the CA1 region of the dorsal hippocampus by means of a movable microelectrode in normal behaving rats. Large amplitude (less than 4 mV) negative EEG spikes (SPKs) of 40-100 msec duration with frequencies in the range of 0.2-5/sec were consistently recorded from the middle apical dendritic layer (stratum radiatum) during awake immobility, grooming and slow-wave sleep. SPKs were replaced by rhythmical slow activity (RSA) during walking and paradoxical sleep. Laminar analysis indicated that SPKs were positive in stratum oriens, negative in stratum radiatum and polarity reversal just below stratum pyramidale. Peak positivity (about 1 mV on average) and peak negativity (2 mV) occurred some 80 micron above and 200 micron below the reversal point, respectively. The SPKs were invariably accompanied by synchronous burst discharges in stratum pyramidale. Bilateral recordings demonstrated the SPKs occurred synchronously in large areas of the CA1 field of the two hippocampi. These results suggest that the SPK represents a massive synaptic excitation of middle apical dendrites triggering synchronous burst discharges in a population of pyramidal cell bodies. A possibility was discussed that these non-pathological SPKs and interictal spikes share some common underlying mechanisms.

Reticular formation neurons related to tongue movement in the behaving cat.

We have found a number of cells related to tongue movement in the medial brain stem reticular formation of the unanesthetized cat. These cells constituted less than 2% of the cells tested in this region and were distributed throughout several nuclei in the medulla and pons including nucleus reticularis pontis caudalis, nucleus reticularis gigantocellularis, and the border between nucleus reticularis paramedianus and nucleus interfascicularis hypoglossi. All observed tongue movement cells (N = 6) fired maximally during protrusive tongue movements. One medullary cell discharged primarily during the protrusive tongue movement to the ipsilateral side, whereas no lateral preference was detected in the other cells. Gustatory and mechanosensory stimulation of the tongue was unnecessary for inducing discharge in these cells. Tongue movement-related cells shared several characteristics that differentiated them from adjacent reticular formation cells, including absence of response to startle-inducing auditory stimuli and low levels of spontaneous waking and sleep activity. In two pontine cells located near the trigeminal motor nucleus, spike-triggered averages of tongue EMG revealed a short-latency (5 ms) inhibitory effect on the ipsilateral genioglossus muscle by the units' discharge. We suggest that neurons of this type might be involved in tongue-jaw coordination during mastication, licking, and grooming.

Single-cell activity and synchronous bursting in the rat hippocampus during waking behavior and sleep.

Eighty-one of eighty-five isolated neurons recorded from the rat dorsal hippocampus (Ammon's horn and area dentata) were classifiable into four types according to their firing patterns and behavioral correlates. Sixty-two percent of the total neurons were complex spike cells which fired slowly (less than 2/s) most of the time, but were most active during slow-wave sleep, slightly less active during awake immobility, and least active during awake movement and paradoxical sleep. Theta cells (21% of the total) generally fired much faster than complex spike cells and increased their discharge rate whenever rhythmical slow activity was present in the hippocampal EEG (i.e., during awake movement and paradoxical sleep). The behavioral correlates of seven putative granule cells were similar to those of theta cells. However, the behavior-dependent changes in firing frequency were much more pronounced in the former than in the latter. Three (fast) complex spike cells were observed which fired much faster than any other complex spike cell during all states and showed the behavioral correlates similar to those of theta cells. The complex spike cells, theta cells, and fast complex spike cells were found in both Ammon's horn and dentate hilus. A type of multiunit burst was recorded from the pyramidal cell layer. It occurred most frequently during slow-wave sleep, slightly less frequently during awake immobility, and was virtually absent during awake movement and paradoxical sleep. The multiunit burst was considered to represent a summed activity of synchronously bursting complex spike cells. Possible cellular mechanisms of the state-dependent activities in the different neuronal populations of the hippocampus are discussed.

Burst characteristics of hippocampal complex spike cells in the awake rat.

Burst firing of hippocampal complex spike cells was examined in the awake rat. The results indicated that each complex spike cell tended to show a unique distribution of the number of spikes in each burst. This distribution was stable over time. The mean number of spikes per burst in 21 cells ranged from 1.06 to 4.07. Each subdivision of the hippocampus (CA1, CA3, dentate) contained cells with a wide range of burst distributions.