Hypertonic urea: Its effects on cortical and subcortical evoked potentials in cat

362

BRAIN RESEARCH

H Y P E R T O N I C UREA: ITS EFFECTS ON CORTICAL A N D SUBCORTICAL EVOKED POTENTIALS IN CAT

ANSELMO PINEDA AND W. ROSS ADEY

Departments of Anatomy and Physiology and the Brain Research Institute, University of California, Los Angeles and the Veterans Administration Hospital, Long Beach, Calif. (U.S.A.) (Received July 22nd, 1966)

Hypertonic urea solution is widely accepted as an agent for the reduction of brain volume and cerebrospinal fluid pressure. This acceptance is the result of the clinical trials of Javid et al. 1°,12,13. Elucidation of its mode of action has been attempted in numerous previous studies. They confirmed the existence of a high osmotic gradient between plasma and brain, which supl:otts the contention that the action of the urea is not dependent upon a diuretic effect11. Reed and Woodbury 2s were the first to suggest a more precise osmotic mechanism. They indicated that the maintenance of a slow gradient for urea across the glial cell membrane resulted in the prolonged reduction in brain water content and presumably brain volume. With these pronounced osmotic cellular changes it might be expected that detectable alterations in the brain electrical activity would occur. In spite of the great interest aroused by hypertonic urea, no previous studies were found relating to possible electrophysiological changes induced in the normal central nervous system. The present paper deals with the effect in the cat of intravenous hypertonic urea on spontaneous and evoked electrical activity in midbrain reticular formation and in cerebral and cerebellar cortices. MATERIAL AND METHODS

Forty adult cats of both sexes and weighing between 1.5 to 3.2 kg were used. All surgical procedures were done under ether anesthesia followed by local infiltration of procaine solution to pressure points repeated at intervals. All animals had tracheotomies and were attached to a Harvard pump respirator. At the conclusion of surgical procedures, ether anesthesia was discontinued and the animals immobilized with intravenous injections of gallamine triethiodide (Flaxedil). Artificial respiration was adjusted in relation to end-tidal CO2 levels. Limited craniectomies of the anterior and posterior fossae were performed and the dura was opened. Evoked potentials in sensorimotor cortex (gyrus proreus), deep brain structures (midbrain reticular formation), and cerebellar cortex (superior vermis) Brain Research, 3 (1966/1967)362-373

EFFECTS OF UREA ON EVOKED POTENTIALS

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were recorded with bipolar and/or monopolar silver leads during cerebral dehydration induced with urea. Midbrain reticular activity was recorded with coaxial electrodes. Exposed brain surfaces were kept moist with silicone oil. Electroencephalographic activity and evoked potentials were recorded in some animals using stainless steel needle electrodes driven through the skull. Single shocks were given at 2-sec intervals to the contralateral sciatic or radial nerves with a Grass stimulator through an isolation unit. The usual stimulating parameters were 5 V and 1.0 msec duration with a peak current density of 1.0 mA. Previous studies with chronically implanted electrodes on the sciatic nerve have indicated that these stimuli are not associated with behavioral responses indicative of pain in the conscious unrestrained cat 1~. Electrical activity was recorded with a Tektronix cathode ray oscilloscope (TyFe 502) and a Grass eight channel electroencephalograph. Responses to 50 stimuli were averaged with a Mnemotron C A T computer. Rectal temperature was maintained at 37 ° during the entire experiment. Hypertonic urea solution 30 ~ (w/v) in a dose of 1 to 1.5 g/kg was slowly injected intravenously during a period of 5 min. The usual brain shrinkage was seen after 15-20 min. Histological checks were made in all experiments involving subcortical electrode placement. RESULTS

(1) Changes in EEG after urea The characteristic E E G pattern in the sensorimotor cortex during immobilization with Flaxedil was variable, with long periods of 'aroused', low-voltage records, alternating with occasional episodes of 'spindling' and extreme pupillary contraction,

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Fig. 1. EEG records from bipolar leads, using silver balls spaced 5 mm for localized cortical recording in gyrus proreus (top trace), and recorded with stainless steel needle electrodes symmetrically placed through skull for biparietal records (lower trace). After urea, trains of spindles appeared in the resting animal (A), but were abolished during sciatic nerve stimulation every 2 seconds (B).

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A. P I N E D A A N D W . R. A D E Y

a manifestation o f behavioral sleep from which the animal could be aroused by light paw pressure. These aspects of the E E G are mentioned in detail in the light of the findings after urea. Whereas the normal animal showed an aroused cortical pattern almost without exception for the first 4 to 5 h o f the experiment, administration o f urea (1.5 g/kg) at this time was followed within 10 to 20 min by increased spindling episodes (Fig. 1). These were not completely symmetrical on the two hemispheres in individual spindle outlines, although they tended to occur simultaneously. Moreover, the incidence of spindles was effectively reduced after urea by auditory stimuli, or stimulation of the sciatic nerve, as described above (Fig. 2). E.E.G. Control

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Fig. 2. Effects ofintercurrent stimulation on EEG changes induced by urea. In each pair of traces, lead derivations were as in Fig. 1. Sciatic nerve stimulation, or brief auditory stimuli abolished spindle trains induced by urea. Presented graphically (lower right), control records for 1 h before urea showed spindling rates of 0 to 2 per rain. However, brief periods of auditory stimulation or sciatic stimulation (S) led to a transient concurrent inhibition of spindling.

These effects of sciatic nerve stimulation were transient, with reversion to the enhanced spindle pattern immediately after stimulation.

(2) Effects of urea on cortical evoked potentials Changes in evoked potentials recorded either monopolarly or bipolarly from the gyrus proreus began 10 to 20 min after urea. In m o n o p o l a r records (Fig. 3), an initial small positive c o m p o n e n t with a typical latency of 15 msec was often little affected B r a i n Research, 3 (1966/1967) 3 6 2 - 3 7 3

EFFECTS QF UREA ON EVOKEDPOTENTIALS

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Fig. 3. Computed averages of 50 cortical potentials evoked by sciatic nerve stimulation recorded monopolarly from gyrus proreus following urea (1.5 g/kg). An initial small positive component (upward deflection)remained essentially unaltered. The succeeding negative and later positive deflections were much augmented. Figures on ordinates indicate elapsed time in minutes after urea administration. Arrow in this and subsequent figures indicates stimulus artefact.

This early positivity was succeeded by a large negativity which was often much augmented, as also were succeeding positive components. This early enhancement ran parallel with the onset of shrinkage in the hemisphere, and was maximal after about 1 h. It persisted for 5 to 7 h in most cases, but sometimes returned to control levels in as short a period as 2 h. The latency to peak of the negative second component of the evoked potential was 30 to 50 msec, and it frequently appeared after urea when virtually absent from preceding control records (Fig. 4). No modification in latency of this negative component occurred during development of enhanced amplitude after urea. The return of the evoked potential configuration to that of controls was examined from the point of view of reversing cortical shrinkage. It appeared that resumption of the original cortical volume was not essential for return to baseline amplitudes. Rather, the cortical volume lagged substantially behind the evoked potential in this Brain Research, 3 (1966/1967) 362-373

366

A. P I N E D A A N D Wo R . ADEY

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Fig. 4. Computed averages of 50 evoked potentials from sciatic nerve stimulation before and after urea (1.5 g/kg). In this case, the negative second component appearing with a latency of 40 msec was small before urea, but became prominent 25 rain after.

regard. It may therefore be assumed that factors other than a mere reduction in volume of cortical elements underly the effects of urea, and the hysteretic aspects of this phenomenon will be discussed below. Observations were also made to exclude as far as possible concomitant effects of cooling and dehydration in the exposed cortex. As mentioned above, the exposed area of the cortex was kept at minimal dimensions and bathed in silicone oil. However, additional observations were made with the bone intact, and appeared to establish amplitude enhancement as a phenomenon directly attributable to urea. With biparietal recording through the intact skull (Fig. 5), an enhancement of secondary and tertiary components of the evoked potentials was observed, with similar times of onset after urea, and similar latencies of individual response components, to those seen with exposed cortex. Brain Research, 3 (1966/1967) 362-373

EFFECTSOF UREA ON EVOKEDPOTENTIALS

367

BIPARIETAL CORTICAL RECORDING

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(3) Effects of urea on evoked potentials in midbrain reticular formation Evoked potentials in the rostral midbrain reticular formation following sciatic nerve stimulation appeared with a latency of 5 to 10 msec, and, with bipolar electrodes, showing a typically single, large early deflection, followed by two or more smaller deflections with latencies of 30 to 50 msec (Fig. 6). As in cortical records, the primary deflection was augmented in computed averages after urea, beginning about 15 min after administration, with augmentation lasting 2 to 3 h. In a comparison of simultaneous averages of cortical and reticular evoked potentials (Fig. 6), it will be seen that augmentation of both potentials began simultaneously about 15 min after the drug was administered and principally involved components having a similar latency of approximately 30 msec. The latency of these components remained essentially unchanged throughout the test period. However, whereas the cortical effect declined after approximately 70 min, the reticular responses Brain Research, 3 (1966/1967) 362-373

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A. PINEDA AND W. R. ADEY

RETICULARFORMATION Control

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Fig. 6. Simultaneously recorded midbrain reticular (left) and cortical potentials (right)evoked by sciatic nerve stimulation, and presented as computed averages of 50 traces. Both leads showed augmented components beginning about 15 min after urea, but declining more rapidly in cortical structures (see text).

remained augmented for more than 100 min. These findings suggest that the cortical shift occurred independently of any ascending reticular influence, and thus requires consideration of local mechanisms in its production.

(4) Effects of urea on cerebellar evoked potentials Despite careful attention to cortical surface temperature, prevention of dehydl ation, and control of carbon dioxide excretion, effects of urea in the cerebellum were less obvious than in the cerebral cortex. Augmentation induced in cerebellar evoked potentials in some cases involved secondary and tertiary components of the response and slight lengthening of latency of the primary deflection (Fig. 7). The cerebellar response was more clearly triphasic in monopolar records, with an initial positivity, and later, longer lasting negative and positive components. Both of the latter phases of the response were augmented after urea (Fig. 8). The time course of the cerebellar augmentation was similar to that in the cerebral cortex.

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369

EFFECTS OF UREA ON EVOKED POTENTIALS

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Brain Research, 3 (1966/1967) 362-373

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A. P I N E D A A N D W . R . ADEY

DISCUSSION

The evoked potential changes observed in this study invite consideration of possible effects arising in synaptic relays in subcortical structures with enhancement of either specific or nonspecific pathways; the local effects of urea on fluid distribution within and between cerebral tissue compartments, including neuronal, neuroglial and intercellular spaces; and as a result of fluid shifts so induced, possible direct effects on focal synaptic potentials occurring in cortical and subcortical neurons (and believed to underlie the polyphasic phenomena of evoked potentials); or on the volume-conducted components of evoked potentials appearing at the cortical surface. Our findings do not suggest that such regions as the rostral midbrain reticular formation, which are able to powerfully influence cortical excitability and the configuration of evoked potentials 2,9,19, are directly responsible for the augmentation following urea. Although midbrain reticular responses were indeed augmented after urea, it was found that this change followed an independent and substantially longer time course than the changes induced in cortical potentials. Rather, it appears that the enhanced amplitude was a manifestation of local processes in cortical and subcortical nuclei. In cerebral cortical evoked potentials, extensive analyses have been made of the source-sink relationships by macroelectrode recording in studies of specific evoked potentials, and of nonspecific evoked potentials, such as augmenting and recruiting responses, and local cortical responses3,2z, 29. More recently, these studies have been extended to intracellular events and their possible relationship to particular components of the evoked potential4,6,24,2% Despite certain inconsistencies between these studies, it has become apparent that substantial degrees of correlation can be found between postsynaptic potentials recorded intracellularly, and certain components of the evoked potential. Specific interrelationships with excitatory or inhibitory PSP's are less clear. Nevertheless, it can be stated unequivocally that the evoked potential is related to the occurrence of slow intracellular events, rather than to the summed activity of neuronal firing. In part, at least, additional spatial correlations can be achieved with the site of origin of these potentials in superficially located axodendritic synapses, as opposed to more deeply located axosomatic synapses. A comparable topographic separation of inhibitory events in spinal motoneurons has been postulated as the basis for 'remote inhibition'7,8. In the frame of the present study, cortical evoked potentials typically showed significant augmentation of negative components, following an earlier, smaller positivity. This may be interpreted as evidence that the source-sink relationship affected here was relatively superficially located in the cortex, perhaps involving axodendritic synapses. The restricted 'window' available with intracellular recording on synaptic activation taking place on remote zones of the dendritic tree in cortical 25 and spinal neuronsT, s, does not encourage the view that a more precise delineation of the location of cellular events modified by urea would necessarily be possible with this technique. We may turn at this point to the question of modification of evoked potential configuration from alterations in fluid content of cortical elements. Clear evidence of a modification in volume of cortical neurons during urea-induced dehydration has been overshadowed in some degree in previous histological studies by preoccupation with Brain Research, 3 (1966/1967) 362-373

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changes in neuroglial characteristics is. In a continuing investigation, to be presented in detail elsewhere z2, of the electron micrographic appearance of the cortex dehydrated with urea, we have noted involvement of the neuron, and particularly the dendritic branches directed toward the cortical surface, in a 'clumping' process that draws them into physically closer association. By contrast, most astrocytic elements appeared unmodified, and the presence of synaptic vesicles in neurons, and of mitochondria in both neurons and neuroglia suggest that the clumping did not arise in regional gradients related to imperfect perfusion or inadequate fixative penetration. Moreover, our normal material comparably prepared has met rigid criteria for normal cytology with the electron microscope17. In these circumstances, two important considerations arise in relation to these modified evoked potentials. Alterations in amplitude of the evoked potentials may relate to the incremented conductance in extraneuronal pathways through an extracellular space with a high macromolecular content zl and offering a preferred pathway for current flow in cerebral tissue1, and exquisitely sensitive to modification in its conductance by bivalent cations is, such as calcium. Neuroglial membrane resistance may also be significantly modified under these conditions, although direct evidence on this point is lacking2°. Mere modification of amplitude by such perineuronal effects cannot explain the differential increment in negative components of the evoked potential, as observed here. It is in this respect that one may seek evidence for a more direct modification in synaptic systems close to the cortical surface, on the basis of classic source-sink phenomena. The studies by Rall et s l Y have emphasized the possible structural and functional relation of dendrodendritic contacts in the olfactory bulb. Speculatively, if such mechanisms do, indeed, play a significant role in cortical electrical potentials, the substrates of interaction between neurons clearly demand evaluation in the frame of reduced tissue water. Electrokinetic movements at membrane surfaces during normal synaptic activation, and dependent on fixed membrane charges 5 which are themselves a property of surface macromolecules14, would then play a regional, and even focal role in modification of selected components of a polyphasic evoked potential. SUMMARY

The effects of urea and concomitant cerebral dehydration on potentials evoked in the cerebral cortex, midbrain reticular formation, and cerebellar cortex by somatic stimulation were studied in the cat. Urea (1.5 g/kg i.v.) typically augmented the negative and late positive phases of triphasic positive-negative-positive evoked cerebral cortical responses recorded monopolarly. This change began about 15 min after urea administration. It paralleled the onset of cerebral shrinkage and was maximal after about 1 h. These evoked potential changes persisted for 5 to 7 h in most cases, but sometimes returned to control levels after about 2 h. Resumption of the original cortical volume was not essential for return to baseline amplitudes. Brain Research, 3 (196611967) 362-373

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Changes in cerebral cortical e v o k e d p o t e n t i a l s i n d u c e d by urea were observed in records w i t h u n o p e n e d c r a n i u m . E v o k e d p o t e n t i a l s in m i d b r a i n reticular f o r m a t i o n were similarly c h a n g e d by urea, b u t modifications in m i d b r a i n p o t e n t i a l s persisted longer t h a n those in cerebral e v o k e d potentials. Effects o f urea on cerebellar p o t e n t i a l s were less obvious t h a n in the c e r e b r a l cortex. In the present study, the e n h a n c e d a m p l i t u d e o f cortical evoked p o t e n t i a l s a p pears to arise in local processes a n d to involve source-sink relationships l o c a t e d relatively superficially in the cortex, p e r h a p s involving a x o d e n d r i t i c synapses. The role o f extracellular fluid with a high m a c r o m o l e c u l a r content, and offering a preferred p a t h way for c u r r e n t flow in cerebral tissue, is discussed in r e l a t i o n to the effects o f urea on e v o k e d p o t e n t i a l s , a n d in r e l a t i o n to the observed differential modification o f specific c o m p o n e n t s o f the evoked response. E l e c t r o k i n e t i c m o v e m e n t s at m e m b r a n e surfaces during n o r m a l s y n a p t i c activation, a n d d e p e n d e n t on fixed m e m b r a n e charges in surface m a c r o m o l e c u l e s , m a y play a focal role in m o d i f i c a t i o n o f p o l y p h a s i c e v o k e d potentials. ACKNOWLEDGEMENTS Studies described here were p e r f o r m e d with the s u p p o r t o f the Veterans A d m i n i s t r a t i o n R e s e a r c h P r o g r a m s , N3-63 a n d N4-63, a n d the U.S. A i r F o r c e , Office o f Scientific R e s e a r c h G r a n t , A F ( A F O S R ) 61-81 a n d A F ( A F O S R ) 49-1387. Mr. G. D i l l a r d p r o v i d e d technical assistance at the beginning o f this project.

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