Differences between cytotoxicity in photodynamic therapy using a pulsed laser and a continuous wave laser: study of oxygen consumption and photobleaching.

Oxygen consumption at the targeted site has a significant effect on dosimetry in photodynamic therapy (PDT). However, oxygen consumption in PDT using a pulsed laser as a light source has not been clarified. We therefore investigated the dependence of cytotoxicity on the oxygen consumption and the photosensitizer photobleaching of PDT using a pulsed laser by comparing with that using a continuous wave (CW) laser. Mouse renal carcinoma cells (Renca) were incubated with a second-generation photosensitizer, PAD-S31. The cells were then irradiated with either a 670-nm nanosecond pulsed light from the 3rd harmonics of a Nd:YAG laser-pumped optical parametric oscillator with a peak fluence rate of approximately 1 MW/cm(2) at 30 Hz or a 670-nm CW diode laser with a total light dose of 40 J/cm(2). Regardless of laser source, cytotoxic effects exhibited cumulative dose responses to the photosensitizer ranging from 12 to 96 microg/ml. However, cytotoxic effect of PDT using the pulsed light was significantly less than that using the CW light with the photosensitizer concentrations of 24 and 48 microg/ml under identical fluence rates. During PDT, the cells exposed to the pulsed light consumed oxygen more slowly, resulting in a lower amount of oxygen consumption when compared with PDT using CW light. In accordance with oxygen consumption, the pulsed light induced significantly less photobleaching of the photosensitizer than the CW light did. These results indicate that the efficiency of PDT using pulsed light is less when compared with CW light, probably being related to suppressed oxygen consumption during the pulsed light irradiation.

Synthesis and evaluation of novel 2-oxo-1,2-dihydro-3-quinolinecarboxamide derivatives as potent and selective serotonin 5-HT4 receptor agonists.

A series of 8'-substituted N-(endo-8-azabicyclo[3.2.1]oct-3-yl)-1-isopropyl-2-oxo-1,2-dihydro-3-quinolinecarboxamides were synthesized. The 5-HT4 receptor agonistic activity was evaluated using the isolated guinea pig ileum preparation. Of the compounds synthesized, N-(endo-8-(3-hydroxypropyl)-8-azabicyclo[3.2.1]oct-3-yl)-1-isopropyl-2-oxo-1,2-dihydro-3-quinolinecarboxamide (15a, TS-951) exhibited the most potent serotonin 5-HT4 receptor agonistic activity. This compound had a high affinity for the serotonin 5-HT4 receptor although it had no affinities for other broad spectrum receptors. Furthermore, it remarkably enhanced gastrointestinal motility in conscious fed dogs without unfavorable effects that non-selective serotonin 5-HT4 receptor agonist has. TS-951 may be useful in improving gastrointestinal dysfunction.

Chronic treatment with FK506 increases p70 S6 kinase activity associated with reduced nitric oxide synthase activity in rabbit hearts.

FK506, an immunosuppressant, modulates phosphorylation of nitric oxide (NO) synthase, and induces cardiac hypertrophy in clinical settings. Having recently reported that chronic treatment with an inhibitor of NO synthase induces cardiac hypertrophy associated with the activation of 70-kD S6 kinase (p70S6K), which plays an important role in cardiac hypertrophy by regulating protein synthesis, we investigated the effects of chronic administration of FK506 on NO synthase and p70S6K activities in hearts. Twenty rabbits were divided into four groups: untreated rabbits, those treated with low-dose FK506 (0.10 mg/kg), those treated with medium-dose FK506 (0.20 mg/kg), and those treated with high-dose FK506 (0.40 mg/kg). FK506 was administered intravenously twice a day. After 4 weeks of treatment with FK506, calcium-dependent NO synthase activity in myocardium in the high-dose FK506 group was lower (P < 0.05) than in the untreated group. p70S6K activity in myocardium in the high-dose group was higher (P < 0.05) than in the untreated group. There was a significant (P < 0.05) inverse correlation between NO synthase and p70S6K activities in myocardium. However, the endothelial-dependent vasodilation of aortic rings or plasma levels of NO metabolites during experimental protocols did not differ among the groups studied. These findings suggest that chronic treatment of FK506 activates p70S6K and reduces NO synthase activity in rabbit hearts. Reduced NO synthase and/or activated p70S6K activities in hearts might contribute to the cardiac hypertrophy observed in some patients receiving FK506.

[Long-term potentiation of the neuronal activity in the motor cortex induced by simultaneous stimulation of the thalamus and somatosensory cortex in cats]. / Dlitel'noe povysheniie neironal'noi aktivnosti motornoi kory, vyzvannoe sovmestnym razdrazheniem talamusa i somatosensornoi kory mozga u koshek.

Long-lasting potentiation of the cat motor cortex units induced by tetanic stimulation of the VL + SCx led to an increase of the motor cortex unit discharge rate. The findings suggest that co-activation of cortico-cortical and thalamo-cortical afferents modifies neuronal activity of the motor cortex at the specific site which receives convergent sensory input from the thalamus and the somatosensory cortex.

Neurobiological basis of motor learning in mammals.

Long-term potentiation (LTP) has been proposed as a model of learning and memory. There is still little evidence, however, linking LTP to cognitive processes. We have chosen to study motor learning, first, because it is relatively simpler than cognitive learning and second, because much of the circuitry involved in motor function is already known. In behavioral studies we determined that the sensory cortex is required for the acquisition of new motor skills. Once a skill is acquired, however, the sensory cortex is no longer necessary in the performance of that skill. In electrophysiological experiments we have shown that LTP can be induced in the motor cortex with stimulation of the sensory cortex (SCx) or associativly when stimulation was applied to both SCx and thalamus. We propose that motor learning involves the formation of loop circuits between the motor cortex and the periphery involving the SCx and the thalamus. At first these loop circuits are diffuse, producing contraction of unnecessary muscles, but become specific by producing LTP through practice.

Information processing within the motor cortex. I. Responses of morphologically identified motor cortical cells to stimulation of the somatosensory cortex.

Inputs from the somatosensory cortex to the motor cortex have been proposed to function in learning of motor skills. In an attempt to analyze how these somatosensory inputs were processed in the motor cortex, neurons in the superficial layer of the cat motor cortex were classified into three groups on the basis of synaptic responses elicited by intracortical microstimulation (ICMS) of area 2. ICMS was delivered through seven electrodes implanted in area 2. When ICMS through one of the seven sites produced a response that was greater than 50% of the response produced by stimulating the seven sites at a time, the site was called a "dominant" site. Type I cells were those that had a dominant stimulation site and showed a constant response latency when examined by a double shock test. Type II cells were those that had a dominant site but displayed a variable latency. Type III cells had no dominant site and showed a variable latency. Latency of type I responses was 1.2-2.6 milliseconds, which was much shorter than that of type II and type III responses. Seventy-nine neurons in layers II/III of the motor cortex, which responded to ICMS in area 2, were stained by intracellular injection of biocytin. From the presence of an apical dendrite and rich spines on the dendrites, 23 type I, 21 type II, and 15 type III cells were classified as pyramidal cells. Type II pyramidal cells were located more superficially than type I and type III pyramidal cells. On the basis of the absence or sparseness of dendritic spines, three type I and four type II cells in layers II/III were classified as nonpyramidal cells. These cells consisted of five small multipolar cells in layer II and a large multipolar cell and a small bitufted cell in layer III. The remaining 11 cells were not classified because of insufficient staining. Since type I and type II cells are considered to represent monosynaptic and polysynaptic responses to stimulation of area 2, respectively, information flow from type I cells to more superficially located type II cells is presumed in layers II/III of the motor cortex. Type III responses suggest the presence of a convergent flow of impulses inside of and/or between areas 2 and 4.

Information processing within the motor cortex. II. Intracortical connections between neurons receiving somatosensory cortical input and motor output neurons of the cortex.

Connections between motor cortical neurons receiving somatosensory inputs from area 2 and large pyramidal cells in layer V were examined in the cat via intracellular injection of biocytin and immunohistochemistry of nonphosphorylated neurofilament proteins (npNFP). Biocytin was injected into pyramidal cells in layers II/III of the motor cortex that responded monosynaptically and polysynaptically to microstimulation of the somatosensory cortex and subsequently stained black by the avidin-biotinylated peroxidase complex method with diaminobenzidine (DAB) and nickel. By using a monoclonal antibody SMI-32 and a modified peroxidase-antiperoxidase method with Tris-aminophenyl-methane (TAPM) and p-cresol as a chromogen, pyramidal cells in layers III and V of the motor cortex were stained red for npNFP. In particular, all the large pyramidal cells in layer V, Betz cells, displayed intense npNFP immunoreactivity not only in the perikarya but also in the dendrites. Double staining with DAB/nickel and TAPM/p-cresol showed that biocytin-filled axon varicosities of the pyramidal cells, which were thought to receive monosynaptic inputs from area 2, made contacts with npNFP-positive dendrites in layers I-III around the biocytin-injected cell and in layers V-VI beneath the cell. The present results suggest that the corticocortical input from area 2 to pyramidal cells in layers II/III of the motor cortex is transferred to layer V pyramidal cells, including Betz cells, as well as to neighboring layer II/III pyramidal cells. Since tetanic stimulation of the somatosensory cortex reportedly produces long-term potentiation in layer II/III cells of the motor cortex, it seems reasonable to assume that a given area of the somatosensory cortex can produce a long-lasting change in the activity of a given group of output cells in the motor cortex.

Input-output organization of the rat vibrissal motor cortex.

The afferent and efferent connections of the vibrissal area of the rat motor cortex (VMCx) were investigated by injecting Phaseolus vulgaris leucoagglutinin (PHA-L) or wheat germ agglutinin-horseradish peroxidase into the physiologically defined VMCx. The VMCx formed reciprocal connections with the primary and secondary somatosensory cortex, lateral and ventrolateral orbital cortex, retrosplenial cortex, and perirhinal cortex. These corticocortical afferents originated from cell bodies in layers II-III and V, and some afferents originated from cell bodies in layer VI of the primary sensory cortex. All of the VMCs efferents terminated in layers I and V or layers I-III and V. The VMCx also formed reciprocal connections with the ventrolateral, ventromedial and centrolateral nucleus, the lateral portion of the mediodorsal nucleus and the posterior complex of the thalamus. It projected bilaterally to the caudate putamen, primarily ipsilaterally to the superior colliculus, anterior pretectal nucleus, and pontine nucleus, and mainly contralaterally to the oral part of the spinotrigeminal nucleus and the reticular formation around the facial nerve nucleus. Finally, injections of PHA-L into the superior colliculus demonstrated that this structure projected contralaterally to the lateral part of the facial nerve nucleus. These data suggest that the VMCx plays a key role in sensorimotor integration, through its extensive interconnectivity with numerous brain structures, and may modulate orientation behaviors by relaying processed information to the superior colliculus.

Synaptic proliferation in the motor cortex of adult cats after long-term thalamic stimulation.

1. One of the hypotheses for information storage in the CNS postulates the induction of structural changes in synaptic circuits. This postulate predicts that behavioral experiences produce changes in neural activity that subsequently induce synaptogenesis in the mature CNS. Available data indicate that the establishment of engrams for novel motor acts may involve alterations of synaptic interactions within the primary motor cortex. The present study examines the hypothesis that patterns of synaptic circuitry and of synaptic activation are rearranged after enhanced neural activity in pathways projecting to the motor cortex. 2. Electrodes implanted in the ventroposterolateral (VPL) nucleus of the thalamus were used for long-term stimulation (20 microA, 4 days) of afferents to the motor cortex in freely behaving, adult cats. This stimulation primarily affected corticocortical inputs from the somatosensory cortex (area 2) to area 4 gamma of the motor cortex. Electron microscopy and stereological procedures were used to compare the numerical density (Nv) of various types of synapses in layers II/III of the stimulated (experimental) motor cortex with the Nv of the corresponding synapses in the contralateral (control) hemisphere. 3. Long-term stimulation produced a significant increase (25.6%) in synaptic Nv in experimental motor cortex. This increase was due primarily to an increase in the Nv of asymmetrical synapses with dendritic spines. The numbers of symmetrical synapses, and of asymmetrical synapses with dendritic shafts, were not affected by long-term stimulation. 4. Synaptic active zones [calculated by measuring the lengths of postsynaptic densities (PSDs)] were significantly longer in experimental motor cortex. Lengthening of PSDs occurred selectively in asymmetrical synapses with dendritic shafts (28% increase). 5. The Nv of synapses having perforations in their PSDs (perforated synapses) was significantly higher in experimental hemispheres. Also increased was the incidence of synapse-associated polyribosomes, which are most commonly found at the base of dendritic spines. An increase in the number of perforated synapses and of polyribosomes are both morphological hallmarks of synaptogenesis. 6. The percentages of synapses having different curvatures (i.e., presynaptically concave, convex, or flat) were similar in experimental and in control motor cortex.(ABSTRACT TRUNCATED AT 400 WORDS)

Long-term potentiation of thalamic input to the motor cortex induced by coactivation of thalamocortical and corticocortical afferents.

1. Intracellular recordings were obtained from neurons in the motor cortex (MCx), in which excitatory postsynaptic potentials (EPSPs) were evoked by microstimulation of the somatosensory cortex (SCx) and the ventrolateral nucleus (VL) of the thalamus. The effects of combined tetanic stimulation of SCx and VL on the amplitudes of these EPSPs were studied. 2. Amplitudes of both corticocortical (CC) and thalamocortical (TC) EPSPs were potentiated after combined tetanic stimulation. This potentiation occurred exclusively in neurons that were located in the superficial layers (II/III) and that received direct input from both the SCx and VL, with both inputs synapsing in close proximity to each other. In all cases, the potentiation lasted until the electrode went out of the cell (21 +/- 25 min, mean +/- SD) the longest being 90 min. We therefore refer to this potentiation as long-term potentiation (LTP). 3. Tetanic stimulation of the thalamus only did not produce LTP in neurons receiving direct input from the VL. 4. LTP was not induced in either CC or TC EPSPs in neurons located in layer V and/or in neurons receiving long-latency CC EPSPs. 5. It is concluded that TC input from the VL to the MCx is potentiated only when coactivated with the CC input from the SCx.

Neurons of the pretectal area convey spinal input to the motor thalamus of the cat.

The aim of this study was to corroborate lesioning work (Mackel and Noda 1989), suggesting the pretectal area of the rostral midbrain acts as a relay between the spinal cord and the ventrolateral (VL) nucleus of the thalamus. For this purpose, extracellular recordings were made from neurons in the pretectal area which were antidromically activated by stimulation in the rostral thalamus, particularly in VL. The neurons were tested for input from the dorsal columns of the spinal cord, the dorsal column nuclei, and the ventral quadrant of the spinal cord. Latencies of the antidromic responses ranged between 0.6 and 3.0 ms (median 1.0 ms): no differences in latencies were associated with either location of the neurons in the pretectal area or with the site of their thalamic projection. Orthodromic responses to stimulation of ascending pathways were seen in the majority of neurons throughout the pretectal area sampled. Latencies of orthodromic responses varied considerably, with ranges of 0.9-9 ms, 6-20 ms, and 2.5-20 ms upon stimulating the dorsal column nuclei, dorsal columns, and ventrolateral quadrant, respectively. The shortest-latency responses to stimulation of the dorsal column nuclei or of the ventral quadrant were likely to be monosynaptic. Temporal and spatial facilitation of the responses to ascending input were common. The data show that neurons of the pretectal area are capable of relaying somatosensory input ascending from the spinal cord to the rostral thalamus. It is suggested that the pretectofugal output to VL converges with cerebellar input in VL neurons and becomes incorporated in cerebello-cerebral interactions and, ultimately, the control of movement.

Identification of neurons producing long-term potentiation in the cat motor cortex: intracellular recordings and labeling.

Intracellular, in vivo recordings were used to identify and subsequently to label neurons in the cat motor cortex in which long-term potentiation (LTP) was induced. Thirty-nine motor cortical neurons that produced excitatory postsynaptic potentials (EPSPs) in response to microstimulation in areas 1-2 (SI) or in area 5a (SIII) were studied. Amplitudes of EPSPs produced in response to test stimulation (1 Hz) were recorded before and after tetanic stimulation (200 Hz, 20 seconds). In 25/39 cells (64%), EPSP amplitudes were significantly increased following the tetanic stimulation (65 +/- 51% average increase), and remained at the potentiated level as long as stable recordings could be maintained (20 +/- 18 minutes, maximum = 90 minutes). LTP was induced exclusively in cells that produced monosynaptic EPSPs in response to area 1-2 or area 5a stimulation. Of the 39 analyzed cells, 13 were labeled by intracellular injections of 5% biocytin. Neurons in which LTP was induced included both pyramidal and nonpyramidal cells and were located exclusively in layers II or III of the motor cortex; cells in deeper cortical layers were not potentiated. These findings indicate that various corticocortical inputs can increase the efficacy of synaptic transmission in a subset of motor cortical neurons. We propose that this plasticity in synaptic transmission constitutes one of the bases of motor learning and memory.

Long-term potentiation in the cat somatosensory cortex.

Intracellular, in-vivo recordings were used to identify neurons in the cat somatosensory cortex in which long-term potentiation (LTP) was induced. Amplitudes of EPSPs produced by microstimulation in the motor cortex (area 4 gamma) were recorded before and after tetanic stimulation (200 Hz, 20 s). In 8/13 cells (62%), EPSP amplitudes increased significantly following the tetanic stimulation. LTP was induced exclusively in cells which produced monosynaptic EPSPs. Six of these cells were labeled by intracellular injections of biocytin. All the cells in which LTP was induced were pyramidal neurons, and were located exclusively in layers II or III of the somatosensory cortex; cells in deeper cortical layers were not potentiated. These data substantiate our previous findings demonstrating LTP in corticocortical pathways and suggest that these pathways play an important role in cortical synaptic plasticity.

Long-lasting potentiation of synaptic potentials in the motor cortex produced by stimulation of the sensory cortex in the cat: a basis of motor learning.

A long-lasting increase in the efficiency of synaptic transmission in the central nervous system has been thought to be one of the bases of learning and memory. To explore the possibility that the motor cortex (area 4 gamma) itself is involved in motor learning, the existence of long-term potentiation (LTP) was examined by recording excitatory postsynaptic potentials (EPSPs) from motor cortical neurons. Short tetanic intracortical microstimulation (ICMS) of the somatic sensory cortex produced a marked potentiation of the EPSPs in a small group of motor cortical neurons. The results raised the possibility that the input from the sensory cortex participates in motor learning and retention of the learned motor skills.

Experiments on functional role of peripheral input to motor cortex during voluntary movements in the monkey.

The functional role of sensory input to the motor cortex was studied by interrupting two major input pathways. One was the dorsal column, which sends the input directly through the thalamus to the motor cortex, and the other was the sensory cortex, which transfers its input through association fibers. Removal of the sensory cortex produced very little motor disturbances and the function recovered within a week. Section of the dorsal column produced some motor deficit, but the deficit was not severe and the animals recovered nearly completely within 2 wk. Combination of dorsal column section and sensory cortex removal produced severe motor deficits. These consisted of loss of orientation within extrapersonal space and loss of dexterity of individual fingers. These deficits never recovered within the duration of observation, which lasted 4-5 wk. It is concluded that the direct sensory input from the thalamus plays an important role in the control of voluntary movements, but loss of its function can be compensated by the input from the sensory cortex. The possible neuronal basis for the observed motor deficits is discussed and it is proposed that the sensory input functions by selectively changing the excitability of cortical efferent zones before and during the execution of voluntary movements. Recovery of motor function following dorsal column section occurred in parallel with the recovery of sensory input to the motor cortex. The recovered function and sensory input disappeared again following section of the association fibers from the sensory cortex. Neuronal mechanism for this observation is also discussed.

Peripheral input pathways to the monkey motor cortex.

We have shown (Asanuma et al., 1979c) that the monkey motor cortex receives peripheral somesthetic inputs directly from the thalamus. In the present experiments, we studied the pathways which mediated these inputs by stimulating superficial radial (SR) and deep radial (DR) nerves and recording evoked potentials from the motor and sensory cortices and the following results were obtained: 1. The focus for SR and DR evoked potentials in the sensory cortex was located in a circumscribed small area whereas in the motor cortex, the evoked potentials were distributed in a wide area along the central sulcus including the distal forelimb area. 2. Ablation of the sensory cortex reduced the size, but neither abolished nor changed the latency of the evoked potentials in the motor cortex. 3. Section of dorsal column nearly abolished the evoked potentials in the motor cortex, but only halved their size in the sensory cortex. 4. Section of ventrolateral cervical column including the spinothalamic tract halved the size of evoked potentials in the sensory cortex, but did not change the size in the motor cortex. 5. It is concluded that direct peripheral inputs to the motor cortex are mediated primarily through the dorsal column system whereas the peripheral inputs to the sensory cortex are mediated through both dorsal column and spinothalamic tract.

Projection from area 3a to the motor cortex by neurons activated from group I muscle afferents.

Two receiving areas in the pericruciate cortex are known for inputs from group I muscle afferents of forelimb nerves. One focus is near the postcruciate dimple of area 3a, and the other in the lateral sigmoid gyrus of the motor cortex (area 4gamma). The cortico-cortical projection of area 3a to 4gamma, and the relay by this projection of group I muscle afferent input to the motor cortex were investigated in cats. The following results were obtained. 1. Seventy-four neurons within area 3a were antidromically activated by intracortical microstimulation of the motor cortex. 2. Although excitation evoked by stimulation of group I muscle afferents could be demonstrated for only a few (8 of 48) cortico-cortical neurons in extracellular recordings, due to the methodological limitations discussed, this input evoked EPSPs in 8 of 9 cortico-cortical neurons recorded intracellularly. Therefore, it is likely that the majority of neurons projecting from area 3a to the motor cortex have an excitatory synaptic input from group I afferents. 3. Neurons projecting from area 3a to the motor cortex were most commonly found in cortical layer III, although some were found in layer V. 4. Five of nine pyramidal tract neurons of area 3a had a strong excitatory synaptic input from group I muscle afferents. 5. A new type of pyramidal tract neuron was found which has cortico-cortical axon collaterals connecting the two cytoarchitectonic regions. These various neurons may be part of a feedback system from muscle afferents to the motor cortex.