In vivo evaluation of lateral lipid chain packing in human stratum corneum.

BACKGROUND/AIMS: The matrix of intercellular lipids (ICL) of stratum corneum (SC) plays an important role in the barrier function of SC. It is important to understand the structure of the ICL matrix for dermatology and cosmetic science. Several methods exist for the analysis of the structure; however, it is difficult to conduct these analyses noninvasively. METHODS: We have developed a method for the analysis of the lateral packing of ICL using Raman spectroscopy. As a proof-of-principle experiment, we prepared a human SC sheet sample and analyzed its structure by the proposed method and by a conventional method involving X-ray diffraction. We compared the results of both methods. In addition, we applied the proposed method to living human skin, and we analyzed the lateral packing of ICL of SC taken from three separate body sites. RESULTS: The results of our method corresponded to those of the conventional method. We detected regional differences of ICL lateral packing using our method in vivo. The results indicated that the packing of ICL in SC taken from the forearm and upper arm are more ordered than that taken from the cheek. CONCLUSION: The results verify that our developed method allows the evaluation of the lateral packing of ICL inside the SC layer of the skin in vivo. Using this method, we can detect regional differences of SC samples taken from various body sites.

Deficiency of presenilin-1 increases calcium-dependent vulnerability of neurons to oxidative stress in vitro.

We examined the function of presenilin-1 (PS1) on neuronal resistance to oxidative stress. CNS neurons cultured from PS1-deficient mice exhibited increased vulnerability to H2O2 treatment compared with those from wild-type mice. Antioxidants protected the cultured neurons against the oxidative stress. An intracellular calcium chelator, BAPTA AM, as well as an L-type voltage-dependent calcium channel blocker, nifedipine, rescued the neurons from H2O2-induced death, while an N-type voltage-dependent calcium channel blocker, omega-conotoxin, or calcium release blockers from ER stores, dantrolene and xestospongin C, failed to rescue them. Wild-type and PS1-deficient neurons showed comparable increases of cytoplasmic free calcium levels after exposure to H2O2. Taken together with the data that PS1-deficient neurons exhibited increased vulnerability to glutamate, these findings imply that PS1 confers resistance to oxidative stress on neurons in calcium-dependent manners.

Dopamine-dependent synaptic plasticity in the striatal cholinergic interneurons.

The striatum, the input stage of the basal ganglia, is a critical brain structure for the learning of stimulus-response habits as well as motor, perceptual, and cognitive skills. Roles of dopamine (DA) and acetylcholine (ACh) in this form of implicit memory have long been considered essential, but the underlying cellular mechanism is still unclear. By means of patch-clamp recordings from corticostriatal slices of the mouse, we studied whether the identified striatal cholinergic interneurons undergo long-term synaptic changes after tetanic stimulation of cortico- and thalamostriatal fibers. Electrical stimulation of the fibers revealed a depolarizing and hyperpolarizing postsynaptic potential in the striatal cholinergic interneurons. The early depolarizing phase was considered to be a cortico/thalamostriatal glutamatergic EPSP, and the hyperpolarizing component was considered to be an intrastriatally evoked GABAergic IPSP. Tetanic stimulation of cortico/thalamostriatal fibers was found to induce simultaneously occurring long-term potentiation (LTP) of the EPSPs as well as the disynaptically mediated IPSPs. The induction of LTP of EPSP required a rise in intracellular Ca(2+) concentration and dopamine D(5), but not D(2) receptor activation. Ca(2+)-permeable AMPA receptors might also play a part in the LTP induction. Blockade of NMDA receptors, metabotropic glutamate receptors, or serotonin receptors had no significant effects. The long-term enhancement of the disynaptic IPSPs was caused by a long-term increase in the occurrence rate but not the amplitude of disynaptically mediated IPSP in the striatal cholinergic interneurons. This dual mechanism of synaptic plasticity may be responsible for the long-term modulation of the cortico/thalamostriatal synaptic transmission.

Dopamine D1-like receptor activation excites rat striatal large aspiny neurons in vitro.

The aim of this study was to elucidate electrophysiologically the actions of dopamine and SKF38393, a D1-like dopamine receptor agonist, on the membrane excitability of striatal large aspiny neurons (cholinergic interneurons). Whole-cell and perforated patch-clamp recordings were made of striatal cholinergic neurons in rat brain slice preparations. Bath application of dopamine (1-100 microM) evoked a depolarization/inward current with an increase, a decrease, or no change in membrane conductance in a dose-dependent manner. This effect was antagonized by SCH23390, a D1-like dopamine receptor antagonist. The current-voltage relationships of the dopamine-induced current determined in 23 cells suggested two conductances. In 10 cells the current reversed at -94 mV, approximately equal to the K+ equilibrium potential (EK); in three cells the I-V curves remained parallel, whereas in 10 cells the current reversed at -42 mV, which suggested an involvement of a cation permeable channel. Change in external K+ concentration shifted the reversal potential as expected for Ek in low Na+ solution. The current observed in 2 mM Ba2+-containing solution reversed at -28 mV. These actions of dopamine were mimicked by application of SKF38393 (1-50 microM) or forskolin (10 microM), an adenylyl cyclase activator, and were blocked by SCH23390 (10 microM) or SQ22536 (300 microM), an inhibitor of adenylyl cyclase. These data indicate, first, that dopamine depolarizes the striatal large aspiny neurons by a D1-mediated suppression of resting K+ conductance and an opening of a nonselective cation channel and, second, that both mechanisms are mediated by an adenylyl cyclase-dependent pathway.

Cholinergic and GABAergic interneurons in the striatum.

In the striatum, interneurons have not been as well characterized physiologically as the spiny projection cells. We found that the neostriatal interneurons can be divided at least into three classes by physiological, chemical and morphological criteria. The first was FS cells (fast-spiking cells) which fired very short-duration action potentials at constant spike frequency during depolarizing pulses, were immunoreactive for parvalbumin (calcium-binding protein), and had axons with very dense collateralization within or near their dendritic fields. Another class was identified as those which fired low-threshold spikes (LTS cells) from hyperpolarized potentials, were positive for somatostatin and nitric oxide synthase (NOS), and had the largest axonal fields. The other class of interneurons had longer-lasting afterhyperpolarizations (LA cells), were positive for choline acetyltransferase, and were mostly large aspiny cells. Glutamic acid decarboxylase (GAD67) or GABA immunoreactivity was detected at the somata or terminals of parvalbumin FS cells and somatostatin/NOS LTS cells, but not of cholinergic LA cells. Substance P, probably released from the collaterals of cells projecting to the substantia nigra, excited LA cells and LTS cells, but not FS cells. These results suggest that the striatum has at least one type of cholinergic and two types of GABAergic interneurons which are different in physiological, chemical and pharmacological characteristics.

Actions of substance P on rat neostriatal neurons in vitro.

Actions of substance P (SP) on the neostriatal neurons in in vitro rat slice preparations were studied via whole-cell patch-clamp recording. Almost all large aspiny neurons (cholinergic cells) and half of the low-threshold spike (LTS) cells (somatostatin/ NOS-positive cells) showed depolarization or an inward shift of the holding currents in response to bath-applied SP in a dose-dependent manner. In contrast, no responses were observed in fast-spiking (FS) cells (parvalbumin-positive cells) and medium spiny cells. Spike discharges followed by slow EPSPs/EPSCs were evoked by intrastriatal electrical stimulation in the large aspiny neurons. Pretreatment with [D-Arg1, D-Pro2, D-Trp7,9, Leu11]-SP, an antagonist of the SP receptor, reversibly suppressed the induction of the slow EPSPs/EPSCs and unmasked slow IPSCs. The SP-induced inward current, although almost unchanged even after the blockade of Ih channels and voltage-dependent Na+, Ca2+, and K+ channels, changed its amplitude according to the Na+ concentration used in both the large aspiny neurons and LTS cells. Thus, the cation current could account for virtually all of the inward current at resting levels in both neurons. These results suggest that the firing of afferent neurons such as striatonigral medium spiny neurons, one of the possible sources of SP, would increase the firing probability of the two types of interneurons of the neostriatum by SP-receptor-mediated opening of tetrodotoxin-insensitive cation channels.

Temporal and spatial characteristics of tonically active neurons of the primate's striatum.

1. Tonically active neurons (TANs) in the primate striatum develop transient responses to sensory conditioning stimuli during behavioral training in classical conditioning tasks. In this study we examined the temporal characteristics of such TAN responses and mapped the sites of TANs responding to auditory and visual conditioned stimuli in the striatum in macaque monkeys. We further mapped the locations of TANs recorded acutely in the squirrel monkey striatum in relation to the neurochemically distinguished striosome and matrix compartments of the striatum, and made quantitative comparisons between the densities and compartmental distributions of TANs and those of four major types of striatal interneuron identified by histochemical and immunohistochemical staining. 2. We made recordings from 858 TANs at different sites in the striatum in two behaving macaque monkeys at different times during training with auditory (click) and visual (light-emitting diode flash) conditioning stimuli. TANs distributed across large parts of the striatum developed responses to the conditioning stimuli. The responses comprised a decrement of tonic firing (pause) followed by a rebound excitation. Measurements were made of the onsets, offsets, and durations of the pauses of individual TANs and of the interspike intervals (ISIs) of the same cells. 3. The mean duration of the pause responses (268.3 ms) was greater than the mean ISI of the same neurons (181 ms), suggesting that the pause represents an active suppression of TAN firing. The coefficient of variation (CV) for the pause responses was 0.28, compared with a CV of 0.63 for the same cells' ISIs. The population CV for the pauses was 0.16, compared with a population CV of 0.20 for the ISIs. These data, together with temporal analysis of the responses and population histograms, suggest that the pauses became temporally aligned across large parts of the striatum after learning. Analyses of variance (ANOVAs) were carried out to determine whether there were differences in the onset and offset latencies of the pause response or in the durations of the pause responses for TANs at different sites. These analyses suggested that, with rare exceptions, there was no difference in the timing of the TAN responses across large (> 10 mm3) parts of the striatum. 4. Comparisons of TAN responses in different regions of the striatum showed that, for responses to a given modality of conditioned stimulus, there were no significant differences in pause offset times for TANs recorded in the caudate nucleus or putamen, or for TANs recorded in more anterior or more posterior parts of these nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)

The basal ganglia and adaptive motor control.

The basal ganglia are neural structures within the motor and cognitive control circuits in the mammalian forebrain and are interconnected with the neocortex by multiple loops. Dysfunction in these parallel loops caused by damage to the striatum results in major defects in voluntary movement, exemplified in Parkinson's disease and Huntington's disease. These parallel loops have a distributed modular architecture resembling local expert architectures of computational learning models. During sensorimotor learning, such distributed networks may be coordinated by widely spaced striatal interneurons that acquire response properties on the basis of experienced reward.

Effect of the nigrostriatal dopamine system on acquired neural responses in the striatum of behaving monkeys.

Dysfunction of the nigrostriatal dopamine system results in marked disorders of movement such as occur in Parkinson's disease. Functions of this dopamine-containing projection system were examined in monkeys trained in a classical conditioning task, and the effects of striatal dopamine depletion were tested. Unilateral dopamine loss substantially reduced the acquired sensory responsiveness of striatal neurons monitored electrophysiologically. This effect was ipsilateral and selective, and could be reversed by apomorphine. These results suggest that the primate nigrostriatal system modulates expression of neuronal response plasticity in the striatum during sensorimotor learning.

Responses of tonically active neurons in the primate's striatum undergo systematic changes during behavioral sensorimotor conditioning.

The basal ganglia have been implicated in motor planning and motor learning. In the study reported here, we directly tested for response plasticity in striatal neurons of macaque monkeys undergoing Pavlovian conditioning. To focus the study, we recorded from the tonically active neurons (TANs) of the striatum, which are known to respond to conditioned sensory stimuli that signal reward delivery and elicit behavioral reactions. The activities of 858 TANs were recorded extracellularly from the striatum in alert behaving macaque monkeys before, during, and after the acquisition of a classical conditioning task. Two monkeys were trained to lick reward juice delivered on a spoon simultaneously with the presentation of a click. Almost no licks were triggered by the cues at the start of training, but by the fifth day more than 90% of licks were triggered, and values were near 100% for the remainder of the 3 week training period. In the striatum, only a small number of TANs responded to the clicks at the start before conditioning (about 17%). During training, the numbers of responding TANs gradually increased, so that by the end of training more than 50-70% of the TANs recorded (51.3-73.5%) became responsive to the clicks. The responses consisted of a pause in firing that occurred approximately 90 msec after the click and that was in some cells preceded by a brief activation and in most cells was followed by a rebound excitation. Prolonged recordings from single TANs (n = 6) showed that individual TANs can acquire a conditioned response within at least as short a time as 10 min. TANs retained such responsiveness after overtraining, and also after a 4 week intermission in training. When the monkey was trained to receive rewards in relation to a new conditioning stimulus, TANs were capable of switching their sensory response to the new stimulus. Histological reconstruction showed that the TANs that became responsive were broadly distributed in the region of striatum explored, which included the dorsal half to two-thirds of the caudate nucleus and putamen over a large anteroposterior span. We conclude that, during the acquisition of a sensorimotor association, TANs widely distributed through the striatum become responsive to sensory stimuli that induce conditioned behavior. This distributed change in activity could serve to modulate the activity of surrounding projection neurons in the striatum engaged in mediating learned behavior.

Activity of primate putamen neurons is selective to the mode of voluntary movement: visually guided, self-initiated or memory-guided.

The aim of this report was to investigate the neural processes of movement initiation and control in which the basal ganglia play an essential role. Single-neuron activity was recorded in the putamen of monkeys performing learned arm movements initiated in three different modes: sensorially guided, internally-timed self-initiated and memory guided. There were no significant differences in the magnitude and timing of both prime mover and supporting muscle activity between the three modes of movement. Over half of the task-related neurons showed strong activity in one of the three modes of movement initiation, but were only slightly activated in the other two modes. No clear preference for a particular movement mode was evident in the population of putamen neurons as a whole. These results are consistent with the hypothesis that there are heterogeneous groups of neurons in the putamen, and that each group of neurons participates in retrieving a different kind of information required for movement based on either external sensory events or on internally stored information.

Age-associated and cell-type specific changes in NGF requirement for neurite regeneration from trigeminal ganglion cells of the shrew (Suncus murinus).

Low density (20-50 cells/cm2), dissociated cultures of trigeminal ganglion (TRG) cells of the shrew, were made in a serum-free medium, and neurite growth was compared across ages of the animal and between NGF-free and NGF-rich conditions. TRG cells from newborn shrew (0-3 days old), which grew long neurites in an NGF-rich medium, failed to grow any neurites in an NGF-free medium. In contrast, TRG cells from aged shrew (16-19 months), which grew neurites without NGF, exhibited no further increment in neurite length when NGF (50-200 ng/ml) was added to the culture medium. TRG cells from adult shrew (4-5 months) were revealed to be a mixture of NGF-dependent and NGF-independent cells. The NGF-dependent cells (20% of the population) had large-sized somata of 24-32 microns diameter (L-type cells) and 2-7 long neurites enriched with arborizations. The remaining NGF-independent cells (80%) had small-sized somata (15-25 microns, S-type cells), and grew 1-3 neurites with a small number of arborizations. These findings suggest that the primary sensory neuron of the shrew has a cell-type specific critical period in the aging process with respect to the requirement of NGF for neurite promotion.

Suppression of GABA-induced chloride current by 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2).

The effect of a convulsive agent, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), on the GABA-induced chloride current (ICl) in dissociated mouse sensory neurons was investigated using the whole cell clamp method. Trp-P-2 reversibly suppressed the GABA-induced ICl in a dose-dependent manner. The IC50 for Trp-P-2 on the ICl evoked by 3 microM GABA was 11.1 microM. Ro15-1788 had no effect on the suppressive action of Trp-P-2. These results suggest that the observed effect of Trp-P-2 is mediated by its action as an antagonist at GABAA receptors.

Modulation of Ca-channel current by an adenosine analog mediated by a GTP-binding protein in chick sensory neurons.

Inhibitory modulation of the high-voltage-activated (HVA) Ca-channel current by 2-chloroadenosine (2CA) was studied in chick sensory neurons using the whole-cell clamp method. 2CA reduced the omega CTX-sensitive HVA-current (Aosaki and Kasai 1989) in a dose-dependent manner with a Kd of 0.8 microM. The inhibition by 2CA was also voltage-dependent, being maximal at hyperpolarized potentials, and completely removed at potentials more positive than 30 mV. This voltage-dependence of 2CA action was also evident as a progressive increase in Ca-channel current magnitude during a depolarization which could be described by a single exponential function and which became faster at larger depolarizations. The concentration of 2CA affected the steady-state reduction in Ca-channel current, but did not alter the time-course of current increase during depolarization. The voltage-dependent effect of 2CA was mimicked by intracellular application of GTP-gamma S, but not by phorbol ester, arachidonic acid or nitroprusside. These results are consistent with model in which 2CA activates a G-protein, which then unmasks an additional activation gate on the Ca-channel.

Characterization of two kinds of high-voltage-activated Ca-channel currents in chick sensory neurons. Differential sensitivity to dihydropyridines and omega-conotoxin GVIA.

High-voltage-activated (HVA) Ca-channel currents in chick sensory neurons were characterized by dihydropyridine compounds (DHPs) and omega-conotoxin GVIA (omega CTX) using patch-clamp methods. In single-channel recordings, two HVA-currents were identified by their single-channel conductances, 13 pS and 25 pS in 110 mM BaCl2. DHPs selectively affected the large-conductance channel. omega CTX (5 microM), on the other hand, irreversibly eliminated only the small-conductance channel, while the large-conductance channel was either unaffected or only transiently blocked. In whole-cell recordings the macroscopic HVA-current was completely and irreversibly blocked by omega CTX but insensitive to DHPs in 60% of the cells. This current presumably was carried by the 13 pS channel. In the remaining cells, a part of the HVA-current (10%, SD = 11%) was either unaffected or transiently blocked by omega CTX and was sensitive to DHPs. This current presumably was carried by the 25 pS channel. Inactivation of both macroscopic current component was incomplete during a 150 ms long depolarization. Our data suggest that the HVA-currents in chick sensory neurons are carried by two distinct Ca-channels that are differentially affected by omega CTX and DHPs.

Divalent cation dependent inactivation of the high-voltage-activated Ca-channel current in chick sensory neurons.

We have used the whole-cell clamp technique to investigate inactivation of the omega-conotoxin sensitive high-voltage-activated Ca-channel current (HVA current [2]) carried either by Ca, Ba or Sr (2.5 mM) in chick sensory neurons. At a low internal EGTA concentration (0.1 mM), Ca-channel currents clearly inactivated irrespective of the species of divalent cation carrying the current. During 150 ms pulses, current inactivated to 0.57, 0.67 and 0.75 of the peak current in Ca, Ba and Sr solution, respectively. Time constants of inactivation (26 +/- 10 ms and 280 +/- 50 ms, mean +/- S.D., in Ba) were largely independent of the membrane potential. Double-pulse experiments showed that the amount of inactivation left by a pre-pulse was proportional to the amplitude of the current evoked by the pre-pulse. No inactivation was induced by an outward current elicited by a strong depolarization to +60 mV. With an internal EGTA concentration of 20 mM, the amount of inactivation was significantly smaller. In conclusion, the inactivation of the HVA Ca-channel currents during current flow depends mostly on the entry of divalent cations irrespective of their species.

Presynaptic Ca-antagonist omega-conotoxin irreversibly blocks N-type Ca-channels in chick sensory neurons.

The present studies on electrophysiological and pharmacological differences of the three types of Ca-currents (N-, L- and T-types) in whole-cell clamped, cultured embryonic chick sensory neurons revealed that the majority (94%) of the Ca-currents in the nerve cells were the N-type, omega-Conotoxin (omega CTX, 5 microM), a blocker of transmitter release at the presynaptic terminals, induced a complete and irreversible blockage of Ca-currents elicited from the resting membrane potential (-60 mV) in 29 cells among 58. The Ca-currents thus irreversibly blocked by the omega CTX were determined as the N-type (neuronal), as they were insensitive to nifedipine (5 microM) or were reduced in amplitude by Bay K 8644 (5 microM). A small fraction (12%) of the total Ca-currents, which were still present after the omega CTX treatment (in the rest of 29 cells), were pure L-type (long-lasting) Ca-currents, as they were enhanced by the Bay K and were blocked by the nifedipine. omega CTX was a partial and reversible blocker of the L-type Ca-currents. Furthermore, T-type (transient) Ca-currents elicited in the hyperpolarized membrane (at -100 mV) were blocked by omega CTX in an incomplete and reversible manner. The N-type Ca-currents thus separated in the nerve cells exhibited various differences in features of the voltage-dependence and ionic selectivity from the L- and T-type Ca-currents.