Katsuei Shibuki

Dynamic imaging of somatosensory cortical activity in the rat visualized by flavoprotein autofluorescence

We used autofluorescence of mitochondrial flavoproteins to image cortical neural activity in the rat. Green autofluorescence in blue light was examined in slices obtained from rat cerebral cortex. About half of the basal autofluorescence was modulated by the presence or absence of O2 or glucose in the medium. Repetitive electrical stimulation at 20 Hz for 1 s produced a localized fluorescence increase in the slices. The amplitude of the increase was 27 ± 2 % (mean ±s.d., n= 35). Tetrodotoxin or diphenyleneiodonium, an inhibitor of flavoproteins, blocked the autofluorescence responses. The autofluorescence responses were not observed in slices perfused with calcium‐, glucose‐ or O2‐free medium. In the primary somatosensory cortex of rats anaesthetized with urethane (1.5 g kg−1, i.p.), an activity‐dependent increase in autofluorescence of 20 ± 4 % (n= 6) was observed after electrical cortical stimulation at 100 Hz for 1 s, and an increase of 2.6 ± 0.5 % (n= 33) after vibratory skin stimulation at 50 Hz for 1 s applied to the plantar hindpaw. These responses were large enough to allow visualization of the neural activity without having to average a number of trials. The distribution of the fluorescence responses after electrical or vibratory skin stimulation was comparable to that of the cortical field potentials in the same rats. The fluorescence responses were followed by an increase in arterial blood flow. The former were resistant to an inhibitor of nitric oxide synthase, while the latter was inhibited. Thus, activity‐dependent changes in the autofluorescence of flavoproteins are useful for functional brain imaging in vivo.

DYNAMIC PROPERTIES OF NITRIC OXIDE RELEASE FROM PARALLEL FIBRES IN RAT CEREBELLAR SLICES

1. Nitric oxide (NO) release following repetitive electrical stimulation was studied in the molecular layer of rat cerebellar slices using electrochemical NO probes. 2. In parasagittal slices of the vermis, most Purkinje cells showed climbing fibre responses in response to white matter stimulation without accompanying NO release. 3. In frontal slices, parallel fibre volley potentials and NO release were elicited concurrently by parallel fibre stimulation. 4. The NO release following parallel fibre stimulation was not affected by blockers of non‐NMDA, NMDA and metabotropic glutamate receptors. 5. The NO release was reduced significantly (P < 0.001) to 29% of the control level after climbing fibre deafferentation with 3‐acetylpyridine treatment. 6. The rate of NO release was roughly proportional to the second or third power of the stimulus frequency, and to the third power of the extracellular Ca2+ concentration. 7. The rate of NO release was not affected by nicardipine (10 microM). It was reduced to 87 +/‐ 4% (n = 5, mean +/‐ S.E.M.) of the control level by omega‐conotoxin GVIA (0.3 microM), and to 18 +/‐ 4% (n = 4) by omega‐agatoxin IVA (0.3 microM). 8. Tetanic parallel fibre stimulation potentiated NO release by 24 +/‐ 5% (n = 5). 9. These data indicate that NO is derived mainly from parallel fibres. The relationship between NO release and cerebellar synaptic plasticity is discussed.

Layer‐specific NO dependence of long‐term potentiation and biased NO release in layer V in the rat auditory cortex

1 We investigated the role of nitric oxide (NO) in the induction of long‐term potentiation (LTP) in slices prepared from the rat auditory cortex. 2 Tetanic stimulation of layer IV elicited LTP of field potentials in layer II–III (LTPII–III) and in layer V (LTPV). The magnitude of LTPII–III measured at 30 min after tetanic stimulation was 171 ± 9 % (n= 15, mean ± s.e.m.) of the control measured before tetanic stimulation, while that of LTPV was 138 ± 3 % (n= 17). 3 NO synthase (NOS) inhibitors had no apparent effect on LTPII–III, but LTPV was significantly suppressed (P < 0.001). This suppression of LTPV was significantly antagonized by a NO donor (P < 0.001) or a cGMP analogue (P < 0.001). 4 Small non‐pyramidal neurones in the auditory cortex were stained with an anti‐neuronal NOS antibody. More neurones were stained with the antibody in the deeper cortical layers. 5 We measured neocortical NO release with electrochemical NO probes. Layer IV stimulation elicited significantly more NO release in layer V than in layer II–III (P < 0.001). The amplitude of the increase in NO concentration elicited by stimulation at 20 Hz for 5 s was 380 ± 14 pM (n= 55) in layer V and 55 ± 8 pM (n= 5) in layer II–III. 6 NO release in layer V was partially but significantly suppressed by non‐NMDA (P < 0.002) or NMDA (P < 0.002) receptor antagonists. Simultaneous application of the antagonists of the two types blocked NO release almost completely. 7 These results clearly indicate the NO dependence of the induction of LTPV, and the greater NO release in the deeper layer of the rat auditory cortex.

Short-term plasticity visualized with flavoprotein autofluorescence in the somatosensory cortex of anaesthetized rats

In the present study, short‐term plasticity of somatosensory neural responses was investigated using flavoprotein autofluorescence imaging in rats anaesthetized with urethane (1.5 g/kg, i.p.) Somatosensory neural activity was elicited by vibratory skin stimulation (50 Hz for 1 s) applied on the surface of the left plantar hindpaw. Changes in green autofluorescence (λ = 500–550 nm) in blue light (λ = 450–490 nm) were elicited in the right somatosensory cortex. The normalised maximal fluorescence responses (ΔF/F) was 2.0 ± 0.1% (n = 40). After tetanic cortical stimulation (TS), applied at a depth of 1.5–2.0 mm from the cortical surface, the responses elicited by peripheral stimulation were significantly potentiated in both peak amplitude and size of the responsive area (both P < 0.02; Wilcoxon signed rank test). This potentiation was clearly observed in the recording session started 5 min after the cessation of TS, and returned to the control level within 30 min. However, depression of the responses was observed after TS applied at a depth of 0.5 mm. TS‐induced changes in supragranular field potentials in cortical slices showed a similar dependence on the depth of the stimulated sites. When TS was applied on the ipsilateral somatosensory cortex, marked potentiation of the ipsilateral responses and slight potentiation of the contralateral responses to peripheral stimulation were observed after TS, suggesting the involvement of commissural fibers in the changes in the somatosensory brain maps. The present study clearly demonstrates that functional brain imaging using flavoprotein autofluorescence is a useful technique for investigating neural plasticity in vivo.

Sound sequence discrimination learning is dependent on cholinergic inputs to the rat auditory cortex

In rat auditory cortex (AC) slices, synaptic potentiation following heterosynaptic stimulation is affected by the stimulus sequence used for induction. It was hypothesized that this sequence-dependent plasticity might be partly involved in the cellular mechanisms underlying sound sequence discrimination. Sequence dependence is abolished by muscarinic receptor antagonists. Therefore, dependence of sound sequence discrimination learning on cholinergic inputs to the rat AC was investigated. Rats were trained to discriminate the sequences of two sound components and a licking behavior in response to one of two possible sequences was rewarded with water. Atropine, a muscarinic receptor antagonist, attenuated sound sequence discrimination learning. The acquired sound sequence discrimination was not affected by atropine. Injections of the cholinergic immunotoxin 192IgG-saporin into the AC suppressed sound sequence discrimination learning, while discrimination between the two sound components was not affected. An inhibitor of M-current, linopirdine, restores the sequence dependence of synaptic potentiation in the AC slices suppressed by atropine. In this study, sound sequence discrimination learning attenuated by 192IgG-saporin was also restored by linopirdine. These similarities between sequence dependent plasticity in the AC slices and sound sequence discrimination learning support the hypothesis that the former is involved in the cellular mechanisms underlying the latter.

Cerebrospinal fluid nitric oxide metabolites in painful diseases.

To elucidate the involvement of NO in pain transmission in humans, we measured NO metabolites (nitrite/nitrate) in the CSF of patients with painful diseases using an NO analyzer based on the Griess method. The nitrite/nitrate levels in patients with degenerative lumbar disease (DLD), but not those with fracture or appendicitis, were significantly higher than those in an age-matched control group. The duration of pain in the DLD group was much longer than that in the fracture or appendicitis group. The nitrite/nitrate levels in the middle-aged and elderly DLD patients depended on the duration of pain. These data probably suggest that the duration of pain is critical for the elevation in nitrite/nitrate levels.

Anisotropic functional connections between the auditory cortex and area 18a in rat cerebral slices

We developed a new method to visualize the myeloarchitecture in fresh slices, and investigated the properties of the functional neural connections around the boundary between the primary auditory cortex (area 41) and area 18a in rat cerebral slices. A fresh slice illuminated by near-vertical light was observed with a CCD camera. The translucent images of the slice showed contrast patterns very similar to myeloarchitecture. The boundary between these areas was identified by the well-developed layer IV/V in area 41 but not in area 18a. Antidromic/presynaptic components of the field potentials stimulated and recorded across the areal boundary showed symmetric distribution, while the postsynaptic field potentials in the direction from area 41 to 18a were more prominent than those in the opposite direction in layer II/III. In contrast, the dominant direction of propagation of postsynaptic potentials was from area 18a to 41 in layer V. In the presence of 1 microM bicuculline, an inhibitor of GABA(A) receptors, the polysynaptic activities propagating from area 18a into 41 via layer V were elicited by stimulation of area 18a. The propagation measured by Ca(2+) imaging or field potential recordings was potentiated after both areas 18a and 41 were alternately stimulated several times.

Long-lasting enhancement of sound discrimination ability after sound exposure in rats

Changes in the sound discrimination ability of rats were investigated after sound exposure (SE) in a Skinner box. For estimation of the sound discrimination ability, two different amplitude-modulated (AM) sounds (S+ and S-) were presented to the rats deprived of water for 48 h. Pedal press behavior in response to only S+ was rewarded with water. The percentages of trials in which pedal press behavior occurred in response to S+ or S- were calculated separately, and test performance of the rats was determined from the difference between the percentages. Rats were exposed to AM sounds during SE of 48 h, and the sound discrimination test was carried out. Enhancement of discrimination between S+ and S- was elicited by SE in a stimulus-specific manner. Latent extinction of the pedal press behavior in response to sound stimuli was not clearly found after SE. The enhancement of test performance was detected 1-48 h after the cessation of SE, and was blocked by injection of an antagonist of N-methyl-D-aspartate receptors into the auditory cortex bilaterally, immediately before the initiation of SE. These results suggest that SE elicits enhancement of sound discrimination ability, and the responsible site is in the auditory cortex.

Nitric oxide release from substantia gelatinosa of the rat spinal cord in vitro

To study characteristics of nitric oxide (NO) release from substantia gelatinosa (SG) in the spinal cord, we measured NO concentration in transverse spinal cord slices of rats using electrochemical NO probes. Electrical stimulation of the dorsomedial white matter adjacent to SG elicited transient current changes in NO probes placed on SG and the amplitude corresponded to a NO concentration of 200-300 pM. This NO release was not affected by the application of antagonists of glutamate or substance P receptors. The NO release in the rats, which were neonatally treated with capsaicin for denervating C-fibers, was significantly smaller than that in control rats. These data suggest that NO is mainly derived from the unmyelinated afferent nerves in the SG of the spinal cord.

Cerebrospinal fluid nitric oxide metabolites are novel predictors of pain relief in degenerative lumbar diseases.

&NA; This study was undertaken to determine whether or not nitric oxide metabolites (NO2− plus NO3−: NOx levels) in cerebrospinal fluid (CSF) would be predictors of treatment outcome in patients with degenerative lumbar diseases (DLD) including lumbar disc herniation (LDH) and lumbar spinal canal stenosis (LCS). The NOx levels in CSF were measured using an NO analyzer based on the Griess method. Six healthy volunteers and 18 patients with painless diseases were included in the control group. The pre‐ and postoperative NOx levels in 25 DLD patients, who underwent herniotomy for LDH (17 patients) or selective decompression for LCS (eight patients), were analyzed. The postoperative follow‐up periods were approximately 8 months. Nineteen of 25 DLD patients, whose preoperative NOx levels were two standard deviations higher than the mean NOx levels of an age‐matched control group, were included in an NO elevated (NOE) group. Among the 25 DLD patients, the preoperative NOx levels in six patients (young LDH group) were within the normal range. The pain‐related Japanese Orthopaedic Association score and the Hirabayashi recovery rate were respectively used to evaluate the pain severity and the degree of pain relief. The preoperative and changes of postoperative NOx levels in the NOE group were negatively correlated with the Hirabayashi recovery rate. Normal postoperative NOx levels and excellent pain relief were achieved in young DLD patients. In conclusion, the preoperative and changes in postoperative NOx levels are quantitative predictors of postoperative pain relief in DLD patients.