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Human divers exposed to hyperbaric pressure may suffer from cognitive and motor impairments thought to be related to high pressure effects per ce. These effects, termed high pressure neurological syndrome (HPNS), appear at pressure above 1.1 MPa. HPNS involves CNS hyperexcitability that is partially attributed to augmented responses of the glutamatergic N-methyl- d -aspartate receptor (NMDAR). NMDAR is blocked by Mg 2+ (physiologically) and by dl -2-Amino-5-phosphonopentanoic acid (AP5, pharmacologically). We have recently reported that hyperbaric pressure augments rat hippocampus NMDAR synaptic response and generates hyperexcitability. We now test pressure effects on the blockade efficacy of Mg 2+ and AP5. Under high pressure conditions more than double [Mg 2+ ] o and [AP5] o were needed to achieve similar effects on NMDAR synaptic response's amplitude, decay time, and time integral comparable to control conditions. [Mg 2+ ] o and [AP5] o concentration-response curves and the concentration for 50% responses' inhibition (IC 50 s) showed similar normalized pattern at control and pressure for each parameter. We conclude that hyperbaric pressure reduces the efficacy of these NMDAR blockers that may be associated with the receptor conformational change(s). This provides additional mechanism for pressure over activation of NMDAR. Taken together with our previous reports, high pressure modification of NMDAR activity significantly contributes to CNS hyperexcitability and possibly for long term vulnerability.

1. Previous studies have shown that hyperbaric pressure depresses synaptic transmission and have suggested that the effect is primarily on transmitter release. The present study analysed the effects of pressure at a crustacean neuromuscular junction. Changes in pressure were compared to changes in extracellular calcium concentration [Ca2+]o with respect to effects on excitatory junction potential (EJP) amplitude, time course, facilitation and potentiation. 2. The effects of 10.1 MPa pressure on EJP amplitude, facilitation and potentiation, but not time course, were mimicked by reducing [Ca2+]o to approximately one-half the normal level. 3. The effects of pressure and the interaction between compression and calcium concentration were analysed in terms of a model of transmitter release. The model assumes that release is dependent on internal calcium concentration, as modulated by both influx and removal processes; that calcium influx is a saturating function of [Ca2+]o; and that release and removal are saturating functions of [Ca2+]i. 4. The results were consistent with the hypothesis that increased pressure acts primarily to reduce calcium influx into the nerve terminal.

Exposure to high pressures (HP) has been associated with the development of the high pressure neurological syndrome (HPNS) in deep-divers and experimental animals. In contrast, many diving mammals are naturally able to withstand very high pressures. Although at a certain pressure range humans are also able to perform to some extent, the severe signs of HPNS at higher pressures motivated the research on the pathophysiology underlying this syndrome rather than on possible adaptive mechanisms. Thermodynamically, high pressure resembles cooling. Both conditions usually involve reduction in the entropy and slowing down of kinetic rates. We have observed in rat corticohippocampal brain slices that high pressure slows and reduces excitatory synaptic activity. However, this was associated with increased gain of the system, allowing the depressed inputs to elicit regular firing in their target cells. This increased gain was partially mediated by elevated excitability of their dendrites and reduction in the background inhibition. This compensation is efficient at low-medium frequencies. However, it induces abnormal spike reverberation at the high frequency band (50 Hz). Synaptic depression that requires less vesicles/transmitter turn over may serve as an energy-saving mechanism when enzymes and membrane pumps activity are slowed down at pressure. It is even more efficient if a similar reduction is induced in inhibitory synaptic activity. Unfortunately, the frequency response characteristics at this mode of operation may make the system vulnerable to external signals (noise, auditory, visual, etc) at frequencies that elicit 'resonance' responses. Therefore, it is expected that humans exposed to pressures above 1.5 MPa display lethargy and fatigue, certain reduction in cognitive and memory functions when the system is working in an 'economic' mode. The more serious signs of HPNS such as nausea, vomiting, severe tremor, disturbance of motor coordination, and seizures, may be the consequence of an interaction between the 'economic' mode of operation and resonance-inducing environmental disturbances.

Recordings from cerebellar Purkinje cell dendrites have revealed that in response to sustained current injection, the cell firing pattern can move from tonic firing of Ca(2+) spikes to doublet firing and even to quadruplet firing or more complex firing. These firing patterns are not modified substantially if Na(+) currents are blocked. We show that the experimental results can be viewed as a slow transition of the neuronal dynamics through a period-doubling bifurcation. To further support this conclusion and to understand the underlying mechanism that leads to doublet firing, we develop and study a simple, one-compartment model of Purkinje cell dendrite. The neuron can also exhibit quadruplet and chaotic firing patterns that are similar to the firing patterns that some of the Purkinje cells exhibit experimentally. The effects of parameters such as temperature, applied current, and potassium reversal potential in the model resemble their effects in experiments. The model dynamics involve three time scales. Ca(2+)- dependent K(+) currents, with intermediate time scales, are responsible for the appearance of doublet firing, whereas a very slow hyperpolarizing current transfers the neuron from tonic to doublet firing. We use the fast-slow analysis to separate the effects of the three time scales. Fast-slow analysis of the neuronal dynamics, with the activation variable of the very slow, hyperpolarizing current considered as a parameter, reveals that the transitions occurs via a cascade of period-doubling bifurcations of the fast and intermediate subsystem as this slow variable increases. We carry out another analysis, with the Ca(2+) concentration considered as a parameter, to investigate the conditions for the generation of doublet firing in systems with one effective variable with intermediate time scale, in which the rest state of the fast subsystem is terminated by a saddle-node bifurcation. We find that the scenario of period doubling in these systems can occur only if (1) the time scale of the intermediate variable (here, the decay rate of the calcium concentration) is slow enough in comparison with the interspike interval of the tonic firing at the transition but is not too slow and (2) there is a biostability of the fast subsystem of the spike-generating variables.

We studied the effect of olfactory learning on the dendritic spine density of pyramidal neurons in the rat piriform (olfactory) cortex. Rats were trained to distinguish between two pairs of odours in an olfactory discrimination task. Three days after training completion, rats were killed and layer II pyramidal neurons identified by Golgi impregnation were examined with a light microscope. Counts of visible spines were performed along the secondary and tertiary branches of both the apical dendrites and the basal dendrites, which are the sites of intracortical synaptic inputs. An estimate of the true spine density was obtained using Feldman and Peters' method (1979, The Journal of Comparative Neurology, 188, 527--542). The estimated true spine density along apical dendrites was higher in neurons from trained rats than those in pseudotrained and naive rats by 15%. As length of spiny dendrites did not change significantly after learning, the learning-related increase in spine density in neurons from trained rats may indicate on an increased number of excitatory synapses interconnecting pyramidal neurons in the piriform cortex, following olfactory learning.

High pressure induces CNS hyperexcitability while markedly depressing synaptic transmitter release. We studied the effect of pressure (up to 10.1 MPa) on the parallel fibre (PF) synaptic response in biplanar cerebellar slices of adult guinea pigs. Pressure mildly reduced the PF volley amplitude and to a greater extent depressed the excitatory field postsynaptic potential (fPSP). The depression of the PF volley was noted even at supramaximal stimulus intensities, indicating an effect of pressure on the amplitude of the action potential in each axon. Low concentrations of TTX mimicked the effects of pressure on the PF volley without affecting the fPSP. Application omega-conotoxin GVIA (omega-CgTx) reduced the synaptic efficacy by 34.3+/-2.7%. However, in the presence of omega-CgTx the synaptic depression at pressure was significantly reduced. Reduced Ca2+ entry by application of Cd2+ or low [Ca2+]o did not have a similar influence on the effects of pressure. Application of omega-AGA IVA, omega-AGA TK and Funnel-web spider toxin did not affect the synaptic response in concentrations that usually block P-type Ca2+ channels, whilst the N/P/Q-type blocker omega-conotoxin MVIIC reduced the response to 52.7+/-5.0% indicating the involvement of Q-type channels and R-type channels in the non-N-type fraction of Ca2+ entry. The results demonstrate that N-type Ca2+ channels play a crucial role in the induction of PF synaptic depression at pressure. This finding suggests a coherent mechanism for the induction of CNS hyperexcitability at pressure.

Learning-related cellular modifications were studied in the rat piriform cortex. Water-deprived rats were divided to three groups: 'trained' rats were trained in a four-arm maze to discriminate positive cues in pairs of odours, 'control' rats were 'pseudo-trained' by random water rewarding, and 'naive' rats were water-deprived only. In one experimental paradigm, the trained group was exposed to extensive training with rats learning to discriminate between 35 and 50 pairs of odours. Piriform cortex pyramidal neurons from 'trained', 'control' and 'naive' rats did not differ in their passive membrane properties and single spike characteristics. However, the after-hyperpolarizations (AHPs) that follow six-spike trains were reduced after 'extensive training' by 43% and 36% compared with 'control' and 'naive', respectively. This effect was not observed in the piriform cortex of another group of rats, in which hyperexcitability was induced by chemical kindling. In another experimental paradigm rats were trained only until they demonstrated 'rule learning', usually after discriminating between one and two pairs of odours ('mild training'). In this experiment, a smaller, yet significant, reduction (20%) in AHPs was observed. AHP reduction was apparent in most of the sampled neurons. AHP remained reduced up to 3 days after the last training session. 5 days or more after the last training session, AHP amplitude recovered to pre-training value and did not differ between 'trained' rats and the others. Accordingly, training suspension for 5 days or more resulted in slower learning of novel odours. We suggest that increased neuronal excitability, manifested as reduced AHP, is related to the ability of the cortical network to enter a 'learning mode' which creates favourable conditions for enhanced learning capability.

High pressure induces hyperexcitability and convulsions in both intact and decerebrated animals. However, pressure suppresses synaptic transmission in isolated invertebrate preparations. We examined the effect of high pressure on monosynaptic (MSR) and polysynaptic (PSR) reflexes in isolated spinal cords of newborn rats. Reflex activity was recorded extracellularly from the cut ends of the lumbar ventral roots L3-L5 following stimulation of the corresponding dorsal roots. Increasing the stimulus frequency from 0.1 to 2.0 Hz reduced the amplitude of both reflexes by 75%. High pressure (10.1 MPa helium) did not affect this phenomenon. Pressure had no effect on MSR amplitude, but increased PSR amplitude by 30%. For MSR, pressure increased the latency by 25%, duration by 20%, and rise time by 25%. Pressure abolished the moderate (119 +/- 4.5%, mean +/- SEM) posttetanic potentiation observed at 0.1 MPa. For the curve relating MSR amplitude to [Ca2+]o, high pressure produced a slight rightward shift of 0.25 mM without affecting its saturation level. These data suggest that the response of vertebrate central synapses to pressure may be different from previously described invertebrate synapses. Alternatively, if synaptic potentials are reduced at high pressure, other processes that determine excitability must be invoked to account for the relatively stabile reflex response.

Guinea pig cerebrocortical synaptosome preparations were used to study the effect of compression to 62 ATA on 45Ca2+ uptake and [3H]GABA release using a calcium ionophore A 23187, which bypasses the voltage-sensitive calcium channel. Pressure was found to exert a suppressive effect on the A 23187-induced release of [3H]GABA, while having no significant effect on A 23187-stimulated 45Ca2+ uptake. On the other hand, both depolarization-induced 45Ca2+ uptake and [3H]GABA release were inhibited by pressure exposure. These results suggest that pressure may suppress GABA release by affecting pre-synaptic events subsequent to calcium influx.

The effect of reactive oxygen species generated by the interaction of xanthine and xanthine oxidase on synaptic transmission was examined at the squid giant synapse and the lobster neuromuscular junction. Exposure of these synaptic regions to xanthine/xanthine oxidase produced a significant depression in evoked release, with no change in either resting membrane properties or in the action potential. Addition of catalase to the xanthine/xanthine oxidase-containing media partially blocked the synaptic depression, indicating that H2O2 contributes to the synaptic changes induced by exposure to xanthine/xanthine oxidase. H2O2 applied directly to the perfusing media also produced a decrease in synaptic efficacy. The results demonstrate that reactive oxygen species, in general, depress evoked synaptic transmission.

Hyperbaric pressure induces hyperexcitability, tremor, and convulsions in intact animals by mechanisms that are not understood. High pressure induces spontaneous electrical discharges in the spinal ventral roots which we call "tremor-associated activity" (TAA). This study examined the nature of TAA, its likely origins, and its possible contribution in the cervical roots to respiratory difficulties. Activity was recorded extracellularly from the cut cervical ventral roots C1 and C5 in an in vitro brainstem-spinal cord preparation from newborn rats. Under hyperbaric conditions spontaneous TAA was observed either between the typical respiratory bursts in C1 and C5 or immediately after the burst itself. Stimulating the trigeminal nerve at 1 Hz induced a direct excitatory medullospinal reflex which, at high pressure, was followed by a conspicuous TAA. The TAA evoked by the dorsoventral root reflex in isolated spinal cord under hyperbaric conditions was similar to that evoked by the medullospinal reflex. These findings suggest that basic TAA originates at the level of the spinal cord and may be triggered by various synaptic inputs. They further suggest that pressure-induced abnormal activity of the motoneuron pool innervating various respiratory muscles may contribute to respiratory problems encountered under hyperbaric conditions.

1. Propagation of action potentials at high frequency was studied in a branching axon of the lobster by means of simultaneous intracellular recording both before and after the branch point. 2. Although the branching axon studied has a geometrical ratio close to one (perfect impedance matching) conduction across the branch point failed at stimulation frequencies above 30 Hz. 3. The block of conduction after high frequency stimulation occurred at the branch point per se. The parent axon and daughter branches continued to conduct action potentials. 4. Conduction block after high frequency stimulation appeared first in the thicker daughter branch and only later in the thin branch. 5. With high frequency stimulation there was a 10-15% reduction in amplitude of the action potential in the parent axon, a corresponding decrease in the rate of rise of the action potential, a 25-30% decrease in conduction velocity, marked increase in threshold and prolongation of the refractory period. In addition the membrane was depolarized by 1-3 mV. 6. Measurements of the membrane current using the patch clamp technique showed a large decrease in the phase of inward current associated with the action potential, before the branching point. 7. The small membrane depolarization seen after high frequency stimulation is not the sole cause of the conduction block. Imposed prolonged membrane depolarization (8 mV for 120 sec) was insufficient to produce conduction block. 8. In vivo chronic extracellular recordings from the main nerve bundle (which contains the parent axon) and the large daughter branch revealed that: (a) the duration and frequency of trains of action potentials along the axons exceeded those used in the isolated nerve experiments and (b) conduction failure in the large daughter branch could be induced in the whole animal by electrical stimulation of the main branch as in the isolated preparation. 9. Possible mechanisms underlying block of conduction after high frequency stimulation in a branching axon are discussed.

1. Because hyperbaric pressure profoundly depresses excitatory synaptic transmission, it has proved difficult to account for its excitatory effects in the CNS. We tested the hypothesis that hyperbaric pressure might increase excitation by enhancing facilitation and potentiation during repetitive synaptic activation, and/or by selectively depressing inhibitory synaptic transmission. Intracellular microelectrode recordings were obtained from crustacean muscle fibers innervated by single identifiable excitor and inhibitor motor neurons; the preparations were exposed to pressures of 0.1-10.1 MPa. 2. Hyperbaric pressure reduced the amplitude of the singly evoked excitatory junctional potential (EJP), enhanced paired-pulse facilitation, and increased the potentiation elicited by trains of stimuli. The potentiated EJP at 10.1 MPa approached the comparable response evoked at normobaric pressure. 3. Hyperbaric pressure also depressed inhibitory synaptic transmission, measured as depression of the EJP by the inhibitor motor neuron. However, pressure depressed excitatory and inhibitory synaptic transmission to the same extent. Thus there appears to be no selective effect of pressure on the GABA-activated chloride channel. The amplitude of the inhibited EJP at 10.1 MPa remained below that at normobaric pressure, even during repetitive stimulation. 4. The results do not support the hypothesis that pressure increases central excitation by selectively depressing inhibitory transmission per se; enhancement of potentiation, however, probably plays an important role. In this preparation, in which inhibitory transmission also displays facilitation, pressure did not increase overall excitation or alter the balance between excitation and inhibition. 5. These results predict that a pressure-excitable network should encompass excitatory synaptic connections which exhibit pronounced facilitation and inhibitory synapses with little or no facilitation.

1. The ionic mechanisms involved in block of conduction of action potentials following high frequency stimulation were studied in a branching axon of the lobster Panulirus penicillatus. 2. A 2-3 mM increase in extracellular K concentration (normal concentration 12 mM) produced block of conduction into both daughter branches. 3. While conduction block induced by high frequency stimulation occurs first into the large daughter branch and only later into the smaller one, propagation into both branches is blocked simultaneously by increased extracellular K concentration. 4. Increasing extracellular K by 2-3 mM resulted in membrane depolarization, reduction in membrane resistance and reduced excitability. The latter two effects were larger than expected from the small depolarization. It appears that increase of extracellular K has direct effects on membrane excitability. 5. It is suggested that block of conduction after high frequency stimulation results from accumulation of K in the extracellular space. However, in order to account for differential conduction block in the two branches one must assume differential buildup of extracellular K concentration around the two branches during high frequency stimulation. 6. Ultrastructural studies using La and horseradish peroxidase as extracellular markers show that the space around the two branches is similar and is open to the extracellular space. Therefore differences in periaxonal volume cannot account for differential buildup of K around the two branches. 7. It is demonstrated that the lobster axon has a Na+/K+ electrogenic pump. After blocking this pump with ouabain, stimulation at high frequency resulted in a conduction block in the two branches almost at the same time. 8. Injection of Ca2+ intracellularly into the thick branch prevents or delays the appearance of conduction block after high frequency stimulation. 9. A mechanism based on these findings is suggested to explain the differential conduction block seen after high frequency stimulation in a branching axon with almost ideal impedance matching.

The impulse conduction capability of a crustacean bifurcating motor axon was studied under various physiological and physical conditions, and effects of several pharmacological agents were tested. The passive and active membrane properties were measured by intra- and extracellular recording, macropatch clamp and vaseline gap voltage clamp. Block of condition after high-frequency stimulation is caused by accumulation of K+ in the extracellular space, while its differential nature is attributed to early activation of the Na+-K+ electrogenic pump and increased intracellular Ca2+ in the thinner branch. Decreased Na+ and K+ conductance, or increased K+ conductance induced by various drugs, largely reduced the maximal following frequency of the branchpoint. High pressure initially increased the neuron excitability, and later decreased, to below control levels, the axon ability to conduct at high frequency. Cooling had a similar effect on the delayed effects of high pressure. The physiological significance of the frequency-dependent block and its possible role in anesthesia, epilepsy, high-pressure nervous syndrome and behavior are discussed.

The escape reflex of the lobster consists of a series of tail flips resulting from alternating activity of the abdominal flexor and extensor muscles. Electromyographic (EMG) activity was recorded from the medial (DEAM) and the lateral (DEAL1) deep abdominal extensor muscles during free swimming. During the escape response, the muscles were active either synchronously or separately, at frequencies of 100–120 Hz. This activity pattern could be generated either by central programming, or by a peripheral mechanism such as frequency-dependent differential conduction block into one of the two branches of the common excitor axon (C.Ex) innervating these muscles. In order to explore the latter possibility in a living animal, we left the DEAM and DEAL1 muscles innervated only by the C.Ex from the tested segment. This was accomplished by manually cutting all other axons in the nerve under visual control. During escape responses in six successfully dissected animals, we found 27 sudden failures of the DEAM responses and only three in DEAL1. The failures were usually preceded by an increase in the delay of the response. These findings strongly suggest that conduction block occurs in the M branch innervating the DEAM under physiological conditions.

A bifurcating crustacean motor neuron, which serves an integrative function by selectively controlling output to its daughter branches, was examined for its behavioral response to changes in pressure and in temperature when each was varied while the other was held constant, and when both were varied together. The neuron was exposed to helium pressure between 1 and 200 ATA and temperatures between 9 and 22 degrees C. The response of the neuron to pressure changes was biphasic and time dependent. Immediately following a pressure change, action potential amplitude and conduction velocity increased, and the ability of the branch point to pass high frequency trains improved; after 15-20 min at pressures above 35 ATA these measures were depressed below control values. The curve relating functional measures to temperature displayed a time-dependent hysteresis, fast warming leading to values for amplitude, velocity, and branchpoint capacity which corresponded to those made at a point 3-5 degrees C higher during slow cooling. The delayed depressant effects of compression and cooling were synergistic. Low temperature significantly enhanced the effects of pressure on amplitude and conduction velocity; high pressure increased the Q10 of both measures. However, slow cooling antagonized the transient compression-induced excitability increase, and prolonged exposure to hyperbaric pressure diminished the temperature hysteresis. The complex time-dependent changes in this branching axon's ability to conduct are related to previously described changes in membrane potential properties. The responses of this axon to pressure changes are different from responses of other axons studied at hyperbaric pressure. Thus, even within relatively stereotyped axon membrane the effects of pressure are not generalizeable among cells. The possible relevance of these findings to the high pressure nervous syndrome (HPNS) is discussed.

Ciliary beating frequency in tissue culture from frog palate and isolated lung was optically examined using instrumentation that was adjusted to measure a fraction of the surface area of a single ciliary cell. Consecutive 1-s segments of the analogue signal were fast Fourier transformed (FFT) to obtain a power spectrum. At room temperature, these power spectra changed over time from 1 s to the next. Each spectrum contained several dominant frequencies of similar intensities. Cooling the preparation resulted in a single-peak spectrum that was constant over time. A mathematical model is proposed to simulate these findings. The results and the mathematical model support the hypothesis that ciliary beating frequency fluctuates over short periods of time.