Neuronal correlates of an encephalopathy associated with aluminum neurofibrillary degeneration

Brain Research, 97 (1975) 253-264 © Elsevier Scientific Publishing Company, Amsterdam

253 Printed in The Netherlands

N E U R O N A L C O R R E L A T E S OF A N E N C E P H A L O P A T H Y ASSOCIATED WITH ALUMINUM NEUROF1BRILLARY DEGENERATION

D O N A L D R. CRAPPER AND G E O R G E J. T O M K O

Departments of Physiology and Medicine, University of Toronto, Toronto (Canada) (Accepted April 25th, 1975)

SUMMARY

A previous study reported that the intracranial injection of a soluble aluminum salt induced an encephalopathy which may serve as a useful animal model of dementia. An early sign of the encephalopathy in cats was a progressive decrement in both the performance of a short-term visual retention task and acquisition of a conditioned avoidance response in the presence of normal visual discriminationL This study reports that 10 days following the application of aluminum (A1Cla) there was an absence in cat visual cortex of neurons with spontaneous frequencies between 7 and 12 spikes/ sec. The loss was associated with neurofibrillary degeneration and aluminum concentrations in lateral gyrus between 4 and 6 #g/g dry weight. The remaining neurons decreased their variability of response to identical visual stimuli and increased their probability of response and frequency of discharge.

INTRODUCTION

An experimental approach to delineate the operations of a neuronal network is to induce selective neuronal dysfunction and observe the behavior of the resulting network. The discovery that intracranial injection of a soluble aluminum salt causes distinct morphological changes in neurons with large dendrites may provide the necessary tool to accomplish this. In the cerebral neocortex injected A1Cla causes neurofibrillary degeneration primarily in pyramidal shaped cells of layer Ill and V1, 4. Associated with the morphological changes, selective behavioral changes in learning and visual short-term memory have also been reported 3. These observations led to the present study which was undertaken to determine the electrophysiological effects of intra-cerebral injection of a soluble aluminum salt. The spike discharge properties of

254 single neurons from the visual cortex of cats were analyzed during beth spontaneous activity and presentation of a patterned visual stimulus. Similar analyses were performed on a group of neurons from normal cat which served as a control 15. METHODS

Ten days prior to recording cats were prepared by the injection of 7 10 #moles of aluminum chloride at p H 4.0 in a total volume of 0.1 ml under pentobarbital sodium general anesthesia a. Two groups of aluminum-injected (A1) cats were prepared. Five animals received aluminum chloride by infusion 1-2 m m below the cerebral cortical surface in the 1ostral portion of the left lateral gyrus (group 1). Four animals received bilateral injections of the same quantity into the vicinity of the inferior horn of the lateral ventricles and at the same stereotactic coordinates as in a previous study in which cats demonstrated short-term visual retention and acquisition deficits (group 2) a. Comparison of a group of cats injected with 0.1 ml of saline at p H 4.0 to an untreated normal group demonstrated no significant differences in short-term retention and acquisition performance and therefore data on 185 visual cortical neurons obtained from a group of 13 untreated cats reported elsewhere 15 served as a control. On the day of recording the aluminum-treated animals were prepared surgically in a manner identical to the untreated cats. Detailed methods of preparation have been reportedlL Surgery was performed under halothane anesthesia. The visual stimulus, a spatial grating subtending 4 ° of visual arc, was focused upon the area centralis of the right eye. The spatial grating had a frequency of 0.6 cycles/degree, a contrast of 0.5, and an averaged intensity of 1.0 log10 cd./sq.m. Contrast is here defined as, (Lmax -- Lmin)/(Lmax @ Lmin), where L is the stimulus luminance. The grating was of the same orientation and frequency for all experiments and was not optimized for any units. The stimulus was flashed at random intervals between 1 and 20 sec for a duration of 3(30 msec. In addition to paralyzing the animal with gallamine triethiodide (10 mg/kg) the possibility of eye movements was reduced by securing the eyeball with scleral sutures and embedding the globe in a 3 ~ agar solution. The unstimulated eye was covered and the field of vision of the stimulated eye restricted to observation of the stimulus. The background light intensity was 0.4 log10 cd./sq.m. Following surgical procedures the halothane was discontinued and all pressure points and wounds were repeatedly infiltrated with local anesthetic (2 ~ xylocaine). The arterial pressure, pH, Pco2, cardiac rate and cortical surface and body temperatures were monitored and adjusted to maintain a physiological normal preparation. Extracellular unit recordings were made in the left striate cortex with tungsten microelectrodes (Transidyne General Co. No. 4 1 4 : 2 # m tip exposed). A normal E E G 15 was a prerequisite for analysis and neurons which exhibited a significant change in spontaneous mean frequency were excluded. Neuronal spikes were amplitude discriminated and led to a Xerox Sigma 5 computer via an interface for on-line processing 15. A post-stimulus time histogram was generated for each set of stimulus presentations. The following parameters were calculated for each post-stimulus time histogram: the hit probability (PH), defined as the probability of one or more spike

255 discharges within a preselectcd interval of time corresponding to a phasic component of the post-stimulus time histogram. It was calculated during the primary and secondary components of the photic response here referred to as early-on, late-on, early-off and late-off. The change in hit probability with stimulation (APr,) was calculated by subtracting the hit probability during equal interval samples of spontaneous activity from Pr~. The variability coefficient (V) was derived from the equation defining a simple Poisson process 15. It was calculated by measuring the relation between the mean number of spikes/stimulus presentation and the hit probability during the phasic response intervals. With approximately 1130 stimulus presentations, 0.91 < V < 1.09 indicated that the discharge characteristics of the neuron were not significantly different from a Poisson process 15. Following completion of recording the Al-cats were anesthetized with pentobarbital sodium and sacrificed. The hemisphere from which recordings were obtained was fixed in 10 ~o~ formalin for histology1, 4. The contralateral hemisphere was frozen for aluminum assay. Aluminum was determined by an ash method 1° and measurements were made on a Perkin Elmer 305B atomic absorption spectrophotometer with either a flame or carbon furnace atomising unit. RESULTS

(a) Spontaneous activity Comparison of the distribution of spontaneous mean frequencies of neurons in group 1 with the untreated group (Fig. 1A and C) demonstrated a significant decrease in the number of neurons with spontaneous mean frequency between 7 and 10 spikes/sec and an absence of neurons with spontaneous mean frequency above 14/sec. Similarly, in group 2, fe~er neurons with frequencies between 7 and l0 were encountered and neurons above 10/sec were absent (Fig. 1B). In the untreated group 10 ~ of neurons had spontaneous mean frequencies between 7 and 10 and these were located at cortical depths corresponding to supra- and infragranular layers 15. Light microscopic examination of tissue stained by Bielschowsky method from the recording site of the Al-cats revealed that neurons with neurofibrillary degeneration were pyramidal shaped and located mainly in layers IlI and V (see Fig. 4). In this, as in previous studiesl, 4, no cerebral cortical neurons identifiable as granule cells exhibited neurofibrillary degeneration. Recent evidence has indirectly demonstrated that neurons with frequencies between 7 and 12/sec may be pyramidal in shape. Both Pettigrew et al. 14 and Hoffman and Stone s indicated that the majority of neurons with spontaneous mean frequency around 10/sec had complex receptive fields whereas simple or hypercomplex cells had much lower frequencies. Employing an intracellular marker, Kelly and Van Essen found that 23/31 pyramidal shaped cells had complex receptive fields 9. In addition they reported 'that all cells with high spontaneous activity (about 10 ~ of the total sample) were complex cells usually in layer V'. Assuming it is the pyramidal neurons that discharge at high spontaneous frequencies which are susceptible to aluminum, this evidence suggests that the effect is to render them electrically inactive.

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An apparent downward shift was also observed in the distribution of mean frequency for neurons in the Al-cats (Fig. 1A, B, and C). For example, comparison of the percentage of neurons with spontaneous mean frequency less than 2.5 indicated that there were 68 o/and 70 % for the two groups of Al-cats and 58 % for the untreated group*, i.e., 10% less than the Al-cats.

(b) Visual evoked activity DL~chargefrequency. During visual stimulation neurons from the Al-cats responded with significantly higher discharge frequencies. This is illustrated in Fig. 1D and E. In 48 % of neurons from Al-cats the visual stimulus evoked an increase in discharge frequencies of greater than 90 % whereas this occurred in only 20 % of neurons from the untreated group. Moreover, 19 % of the neurons from the untreated group decreased their discharge frequency by more than 10 %; only 8 % exhibited this decrease in the Al-cats. This was corroborated by calculation of the averaged mean frequency * The untreated group contained a considerable n u m b e r of n e u r o n s with s p o n t a n e o u s mean frequencies above 7. F o r valid comparison n e u r o n s with spontaneous m e a n frequency above 7 were eliminated for this calculation.

257 of discharge of neurons with spontaneous mean frequency less than 7.0/sec for 800 msec intervals following the onset of the stimuli and testing for differences. For the Al-cats, the mean was 4.36 ± 4.22 (N = 78), and for the untreated cats it was 2.97 4- 2.35 (N = 91, P < 0.005, Analysis of Variance test). A disorder in inhibitory mechanisms, perhaps analogous to disinhibition, during the stimulus presentation could account for these observations. This is supported by combined intra- and extracellular recordings during the later stages of the aluminum encephalopathy previously reported1, 2. During the transcallosal response neurons recorded during the late encephalopathy and situated in layer III and V lose the long latency IPSP. This is correlated with a decrease in amplitude of the surface recorded negative component of the transcallosal response. A progressive voltage amplitude decline in the late components of the averaged visual evoked potential was also observed and related to the density of subjacent pyramidal neurons with neurofibrillary degeneration. Hit probability analysis. Analysis of the hit probability (PH) demonstrated that A1cats exhibited significantly larger changes in the average hit probability with stimulation, API~, during the primary phasic components of the post-stimulus response (early-on, and early-off, P ~ 0.1301, Table IA). Based on the previous evidence this may be interpreted as phasic disinhibition, that is, there was a loss of inhibition in some neurons during the primary phasic responses. That disinhibition did not occur during spontaneous activity of neurons in the aluminum-treated animals was concluded from the downward shift in the spontaneous frequency distribution histograms and from the observation that the averaged spontaneous mean frequencies of neurons in the Al-cats (1.89 4- 1.63, N = 78) was lower, although not significantly, than in the controls (2.12 4- 1.78, N = 91; for this comparison, neurons with spontaneous mean frequency above 7 were excluded from the control group). In contrast to the primary phasic response the hit probabilities during the secondary phasic component, late-on, were not significantly different (Table IA). An explanation of this may be provided by the observation that in the untreated cats, neurons with spontaneous mean frequencies between 7 and 12 had the smallest APH during the early-on phasic response and the largest during the late-on response 15. The loss of phasic facilitatory input provided by axon collaterals of neurons with spontaneous mean frequency between 7 and 12/sec during the late-on component could have counteracted the disinhibition resulting in the insignificant differences. Response lateneies. Records were obtained from neurons which exhibited unequivocal phasic responses determined from a post-stimulus time histogram and latencies were measured from the onset of the stimulus to the point at which the primary phasic response diverged from background activity. Fig. 2A and B illustrates the relationship for each neuron between the spontaneous mean frequency and latency of the primary phasic response. In the untreated cats the average latency of neurons with spontaneous mean frequencies between 7 and 12/sec, 21.1 ± 2.9 (N = 10), was significantly lower than for neurons with spontaneous mean frequencies below 7/sec, 43.3 ~ 15.5 msec (N = 70, P ~ 0.005). How-

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SPONTANEOUS MEAN FREQUENCY (SPIKES/SEC) F i g . 2. L a t e n c y o f n e u r o n s a s a f u n c t i o n o f t h e i r s p o n t a n e o u s m e a n c o r t e x o f a l u m i n u m t r e a t e d c a t s ( A ) a n d u n t r e a t e d ( c o n t r o l ) c a t s (B).

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ever, differences in latencies of 20 msec or more between the two groups of neurons are much larger than the difference in conduction time between the fast and slow fiber systems from the area centralis to the visual cortex. Recent experiments suggest that the large differences in our preparation may be due to the non-optimal position of the stimulus in the visual field for the majority of neurons. Increases in stimulus response latencies have been measured when the stimulus is delivered remote from the optimal position 16. However, based on the observations of Hoffmann and Stone 8 that complex cells respond with shorter latencies to electrical stinmlation of the visual afferent pathways, this additional evidence does not contradict the argument that cells in the striate cortex of cat with spontaneous mean frequency between 7 and 12/sec may be complex cells. Variability coefficient. In normal cat, measurement of the variability (V) revealed that the spike discharges of many neurons were non-stationary 1~- when compared to a simple Poisson process 15. In comparison, the Al-cats had a considerably smaller percentage of neurons with large variability coefficients, i.e. V > 1.09. This was revealed by measuring the average variability coefficient (V) of all neurons for the 4 phasic responses (Table IB). In the experimental context non-stationarity had been interpreted as preliminary evidence that neurons alter their response characteristics.

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Fig. 3. Distribution of aluminum concentrations (pg/g dry weight) in cerebral cortex in two cats which received bilateral injections of A1Cla into ventral hippocampi (group 2). Assuming the absence of other factors, the observation that neurons with spontaneous mean frequencies between 7 and 12/sec are functionally absent in the A1cats suggest that these neurons may affect the underlying probability of discharge of neurons with lower frequencies. This effect could be mediated by alterations in the inhibitory and facilitory mechanism and reduction in efficacy of these mechanisms could result in a loss of a possible plastic response capability of neurons 15. The observation that neurons with high spontaneous frequency respond with short latencies is consistent with the possibility that these neurons receive visual information early enough to facilitate or modify the response characteristics of other cortical neurons 1~ (see Discussion).

(c) Aluminum concentration and histopathology To establish the neurotoxic concentration for aluminum, atomic absorption measurements were performed on small samples (10-20 mg dry weight) of brain. The average concentration of aluminum from 39 cerebral areas in 6 normal cats was 1.4 ± 0.5 #g/g dry weight 6. The average concentration of aluminum in the lateral and postlateral gyrus for group 2 cats was 5.7 #g/g dry weight (7 samples). The cerebral distribution of aluminum for two Al-cats from group 2, as measured in the contralateral hemisphere from which electrical recordings were made, is shown in Fig. 3. Table II gives the subcortical distribution of aluminum for these brains. Group 2 cats received equal amounts of A1C13 into the region of each hippocampus. These data indicate that aluminum diffuses widely throughout the brain but the uptake varies over a considerable range and some regions even exhibited normal concentrations. The highest concentrations occurred in the regions of injection. The distribution of neurofibrillary degeneration in cerebrum and brain stem was also patchy and was similar to that previously reportedl, 4. In cats of group 2, accumulations of perinuclear and dendritic argyrophilic material was seen in Bielschowsky stained sections from regions in which the aluminum concentration in the contralateral hemisphere exceeded 4 #g/g dry weight. In general, the density of neurofibrillary degeneration in a neocortical region appeared to be approximately proportional to the aluminum concentration above 4 /~g/g. Examples of neurofibrillary degeneration taken from the lateral gyrus are shown in Fig. 4A and B and reveal the highest density of neurofibrillary degeneration to occur in the larger pyramidal shaped neurons of layers III and V.

261 TABLE II ALUMINUM DISTRIBUTIONIN SUBCORTICAL REGIONSFOR CATS A AND B OF FIG-, 3 Asterisk denotes values expressed as #g/ml.

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DISCUSSION

Failure to observe neurons with spontaneous frequencies between 7 and 12/sec in the Al-cats does not constitute p r o o f of their absence. However, at this time the evidence does not contradict the hypothesis that in Al-cats these neurons are electrically dysfunctional, and that the dysfunction is associated with the presence of neurofibrillary degeneration. An explanation of the electrophysiological consequences associated with neurofibrillary degeneration in neurons may be related to the loss of microtubules and subsequent disruption of the cytoplasmic transport mechanism required for the conveyance of trophic substances from soma into dendrites a. Soma synthesized substances are considered necessary for the maintenance of effective synaptic activity and the interval between the appearance of neurofibrillary degeneration and altered electrical activity may be related to the supply and turnover of these substances. Failure of post-synaptic potentials could result in lower spontaneous mean frequency in the affected neurons. Alternatively, loss of subcortical facilitation could also lead to lower spontaneous mean frequency. It is known that aluminum diffuses widely and affects cells in subcortical areas 4. The absence of 7-12/sec neurons in the visual cortex could be the result of a compounded effect of neurofibrillary degeneration in those neurons and the secondary consequence of disfacilitation from subcortical neurons similarly affected. Loss of facilitation is one explanation to account for the observed downward shift in the distribution of spontaneous mean frequency for remaining neurons in the Al-cats. Recurrent axon collaterals of pyramidal shaped neurons of layer l I I and V arborize extensively within the neocortex and give rise to axon terminals morphologically identical to excitatory synapses 11. It is possible that the functional

262

Fig. 4. Neurofibrillary degeneration in cerebral cortical neurons of two group 2 cats: A, rostral lateral gyrus and B, posterior lateral gyrus. Bielschowsky stain. Calibration line: 100 #m.

263 removal of what is probably tonic excitation provided by collaterals from pyramidal shaped neurons with neurofibrillary degeneration resulted in disfacilitation of the remaining neurons. Therefore, we propose the hypothesis that neurons which in the normal cat visual cortex discharge at spontaneous mean frequency between 7 and 12/sec are functionally defective in the Al-cats and that this may be the result of neurofibrillary degeneration. Moreover, indirect evidence indicates the group between 7 and 12/sec would be expected to contain cells which exhibit complex receptive field characteristics. Based on the assumption that the major cytotoxic effect of AI is manifested by neurofibrillary degeneration in pyramidal shaped neurons, the speculation that the axon collaterals of neurons susceptible to neurofibrillary degeneration synapse not only with unaffected pyramidal neurons but also with inhibitory interneurons may explain the observed phasic disfacilitation and disinhibition in Al-cats. Candidate inhibitory interneurons are the pericellular basket cell which form inhibitory axosomatic synapses on many pyramidal cells in the visual cortex 15 and the chandelier cell which form inhibitory axo-dendritic synapses with apical dendrites of pyramidal cells at the border of layer II and II113. This suggests that in normal visual cortex neurons with mean frequencies between 7 and 12/sec act in concert with other afferents to modulate the discharge of neurons directly and via inhibitory interneurons. Functional loss of these neurons could account for not only phasic disinhibition during the primary response but phasic disfacilitation during the late-on response and tonic disfacilitation resulting in lower spontaneous mean frequency. Though disfacilitation and disinhibition could account for the alterations in response characteristics of the remaining neurons, another possibility must be considered. The application of a fluorescent histochemical stain for aluminum, morin v, indicated that aluminum accumulated on the chromatin of most brain cells and was not restricted to those with neurofibrillary degeneration. Therefore, a metabolic effect of aluminum upon neurons which do not develop neurofibrillary degeneration and an associated alteration in excitability cannot be completely excluded at this time. The alterations in unit activity reported here occurred at an early stage in the aluminum encephalopathy during which deficits in the performance of learning and memory tasks occurred, but prior to the onset of focal neurological signs and seizures. Intensive monitoring of unrestrained animals with multiple chronically implanted surface and depth electrodes revealed no abnormal slow wave or seizure activity until the later stages of the encephalopathy 4. The E E G from the lateral gyrus of the animals of this report was normal at the time of unit recording and further supports the assumption that only a small proportion of neurons are primarily affected by the pathological process during the early stage of the encephalopathy. This observation may have an important clinical implication. Disorganization of the E E G in Alzheimer's disease does not correlate well with the clinical state and may not occur until there is moderate to severe dementia 5. Within the limits of the animal experimental model, this study predicts that neurons with neurofibrillary degeneration in Alzheimer's disease may become electrically silent or undergo a reduction in mean spontaneous discharge frequency.

264 The m a j o r finding that n e u r o n s with s p o n t a n e o u s m e a n frequencies between 7 a n d 12/sec are absent also leads to the speculation that these n e u r o n s m a y somehow be involved in the processing or retention of stored i n f o r m a t i o n . T h a t these n e u r o n s can be grouped into a unique class in the cortex is supported by their shorter latencies to response, higher s p o n t a n e o u s m e a n frequencies, pyramidal shape, cortical location a n d the possibility that they exhibit complex receptive fields. T a k e n together with the results of our previous study 15, which d e m o n s t r a t e d that these n e u r o n s also have n o n - s t a t i o n a r y response prolzerties, this leads to the tentative hypothesis that complex cells m a y play a n integral role in processing short-term memories.

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4 5 6 7 8

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15 16

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