Intracellular and extracellular pHs of Streptococcus mutans after addition of acids: loading and efflux of a fluorescent pH indicator in streptococcal cells.

A pH-sensitive fluorescent dye, 2', 7'-bis-(2-carboxyethyl)-5 and 6-carboxyfluorescein (BCECF), was used to determine intracellular pH (pH(in)). The efflux of BCECF loaded into oral streptococcal cells was determined after incubation of the cells at 35 degrees C for 20 min in the presence and absence of glucose. In the absence of glucose, the fluorescence of intracellular BCECF in Streptococcus mutans, Streptococcus sanguis, Streptococcus salivarius and Streptococcus sobrinus decreased only very slightly, indicating that the dye could be useful for pH(in) determination. In the presence of glucose, however, the fluorescence decreased by 57%. Thus, the pH(in) of S. mutans cells was measured by the BCECF method in the absence of glucose at various acidic pH levels by adding lactic, acetic and hydrochloric acids to the cell suspensions. The pH(in) was almost equal to the extracellular pH (pH(out)) for pH(out) values of between 8 and 5, indicating that protons permeated easily across the S. mutans cell membrane. For pH(out) between 5 and 4, pH(in) was constant at around 5, suggesting that the cell membrane was impermeable to protons, or that a cytoplasmic buffering system functioned. pH(in) decreased at pH(out) values of < 4. The constant pH(in) at acidic pH(out) levels could protect intracellular components, such as proteins, against acidification by sugar fermentation.

Effects of conditioning stimulation of the central amygdaloid nucleus on tooth pulp-driven neurons in the cat somatosensory cortex (SI).

To study the limbic control of nociception, we examined the effect of conditioning stimulation of the central amygdaloid nucleus (ACE) on tooth pulp-driven (TPD) neurons in the first somatosensory cortex (SI). Cats were anesthetized with N(2)O-O(2) (2:1) and 0.5% halothane, and immobilized with tubocurarine chloride. The tooth pulp test stimulus was applied by a single rectangular pulse (0.5 ms in duration and 3-5 times the threshold intensity for the jaw-opening reflex). Conditioning stimuli to the ACE consisted of trains of 33 pulses (300 microA) delivered at 330 Hz at intervals of 8-10 s. In 35 out of 61 of the slow (S)-type TPD neurons with latencies of more than 20 ms, conditioning stimulation in the ACE, especially in the medial division, markedly reduced the firing response to the pulpal stimulation. The inhibition of the firing rate in the S-type neurons was 74% of the control. In these S-type neurons, the neurons that were inhibited had significantly longer latencies compared to the non-inhibited neurons (45.0 +/- 17.6 ms, n = 32 vs. 34.8 +/- 10.5 ms, n = 26). In contrast, the ACE conditioning stimulation affected only one out of 18 fast-type TPD neurons with latencies of less than 20 ms. In addition, ACE stimulation had no effect on the spontaneous discharges of either S-type or F-type neurons. The ACE inhibitory effect on S-type neurons was not diminished by naloxone administration (1 mg/kg, I.V. ), while the blockade of histamine H(1)-receptor by diphenhydramine hydrochloride (0.5 mg/kg, I.V.) partially reversed the inhibitory effect. These results suggest that the ACE inhibits ascending nociceptive information to the SI and that this inhibition is mediated in part by histamine (H(1)) receptors. It seems likely that the antinociceptive effect is a neurophysiological basis for stress-induced analgesia (SIA).

Effect of electrical stimulation of the central amygdaloid nucleus on the nociceptive neuron of the cortex (SI) in the cat.

The effect of conditioning stimulation of the central amygdaloid nucleus (ACE) on the response of tooth pulp-driven (TPD) neurons in the first somatosensory cortex (SI) was investigated in cats anesthetized with N2O-O2 (2:1) and 0.5% halothane. The tooth pulp test stimulus was a single 30-450 microA rectangular pulse, and the conditioning stimuli of the ACE were trains of 33 pulses (300 microA) delivered at 330 Hz. The ACE conditioning stimulation markedly suppressed the response of the slow-type neurons with latencies of more than 20 ms without any effect on the discharges of fast-type TPD neurons and spontaneous discharges. This inhibition was 68.9 +/- 24.7% (mean +/- SD) of the control. These findings suggest that there are at least two pathways for the ascending pulpal (nociceptive) information to the SI, and that the ACE modulates the transmission of impulses in one of the pathways.

Tooth pulp stimulation induces c-fos expression in the lateral habenular nucleus of the cat.

Detection of Fos protein expression by the peroxidase-antiperoxidase method was used to determine the area in the habenular (Hb) complex responding to electrical stimulation of the tooth pulp in the cat anaesthetized with pentobarbital. In the anaesthetic-injected group, the Fos-positive neurones were found bilaterally in the lateral Hb nucleus (HbL). Tooth pulp stimulation (intensity: 3 times the threshold for jaw-opening reflex) increased the number of positive neurones within the HbL by up to 300%, but did not induce any expression in the medical Hb nucleus. The increase in HbL was inhibited by morphine (2 mg kg-1, i.p.). These findings and the results of previous research suggest that HbL neurones are involved in defensive mechanisms by means of antinociception following noxious stimulation.

Electrical stimulation of tooth pulp increases the expression of c-fos in the cat supraoptic nucleus but not in the paraventricular nucleus.

Immunoreactivity to Fos protein was detected in the supraoptic (SON) and para-ventricular (PVN) nuclei of the cat using immunohistochemical methods. In the intact animal group, only a few Fos-positive neurons were observed in the PVN, but the SON did not contain any positive neurons. Intraperitoneal injection of pentobarbital sodium (Nembutal: 35 mg/kg) induced c-fos expression in the SON, but not in the PVN. Electrical stimulation of tooth pulp with an intensity that was 3 times the threshold of the jaw-opening reflex (200-600 microA) increased the number of Fos-positive neurons in the SON by up to 388% as compared with those of the Nembutal group, whereas the stimulation did not alter the number in the PVN. The increase was observed throughout the extent of the SON. In addition, morphine treatment (2 mg/kg, i. p.), 5 minutes before tooth pulp stimulation, considerably inhibited the increase in the SON. There were no significant differences among the 3 groups (intact, Nembutal, morphine) in the number of positive neurons in the PVN. These findings suggest that these hypothalamic nuclei have different functional roles and that the SON is involved in nociception and/or the consequent emotional and visceral reactions.

Conditioning stimulation of the central amygdaloid nucleus inhibits the jaw-opening reflex in the cat.

To elucidate a function of the central amygdaloid nucleus (ACE) in the trigeminal system, the ACE conditioning effect on the jaw-opening reflex (JOR) regarded as a nociceptive reflex was investigated in the cat anesthetized with pentobarbital sodium. The JOR to molar tooth pulp stimulation with an intensity 1.2-1.5 times the threshold was recorded in the ipsilateral digastric muscle. As conditioning stimulation, a train of 33 rectangular pulses (0.5 ms in duration) at 330 Hz with an intensity of 300 microA was applied to the ipsilateral ACE. The conditioning stimulation inhibited a JOR that had a latency of 7.90 +/- 0.88 ms (n = 36). The inhibition was maximum (83.1 +/- 11.2%) at a conditioning-test (C-T) interval of 110 ms and continued for C-T intervals of up to 1,000 ms. Likewise, microinjection of 0.5 M monosodium glutamate (10 microliters) into the ACE-inhibited the JOR for approximately 10 min. Additionally, the ACE conditioning stimulation inhibited the JOR induced by the stimulation of the sensory trigeminal nuclear complex in a similar manner, but not the jaw-opening response induced by the stimulation of the trigeminal motor nucleus (Mo V). Also, the conditioning stimulation influenced neither the evoked potentials induced by the tooth pulp stimulation at the main sensory and rostral nuclei nor the jaw-closing reflex induced by the stimulation of the mesencephalic trigeminal nucleus (Mes V). These results suggest that the excitation of the cell bodies in the ACE exerts an inhibitory modulation on the JOR with no effect on the non-nociceptive reflex such as the jaw-closing reflex at the level of Mo V.

Inhibition of jaw-opening reflex by stimulation of the central amygdaloid nucleus in the cat.

The effect of stimulation of the amygdaloid complex on the jaw-opening reflex (JOR) was studied in the cat anesthetized with pentobarbital sodium. The conditioning stimulation of the central amygdaloid nucleus (ACE), but not the other amygdaloid nuclei, markedly inhibited the JOR induced by the tooth pulp stimulation. Ipsilateral ACE conditioning stimulation with 300 microA produced inhibition which lasted approximately 500 ms from the cessation of the stimulation. Additionally, the microinjection of monosodium glutamate into ACE elicited inhibition of the JOR that lasted about 10 min. These findings suggest that the excitation of the cell bodies in ACE exerts inhibitory action on the trigeminal nociceptive reflex.

Inhibitory effect of entopeduncular nucleus on the jaw-opening reflex induced by the tooth pulp stimulation.

The effect of stimulation of the entopeduncular nucleus (EP) on the jaw-opening reflex (JOR) was studied in the cat anesthetized with sodium pentobarbital. JOR was quantified by the digastric electromyogram. The conditioning stimulation (train of 33 pulses at 330 Hz, 0.5 ms duration, 50-400 microA) of the EP powerfully inhibited the JOR induced by the tooth pulp stimulation. Conditioning stimulation with 300 microA produced inhibition which continued approximately 400 ms from the cessation of the stimulation. The injection of monosodium glutamate into EP elicited inhibition of the JOR that lasted about 15 min. Additionally, the conditioning stimulation inhibited the JOR induced by the stimulation of trigeminal rostral and caudal nuclei in a similar manner but not jaw-opening response induced by the stimulation of the trigeminal motor nucleus. Therefore, it is possible that the excitation of the EP exerts inhibitory modulation of JOR at the motor nucleus rather than the trigeminal sensory nucleus.