(Stroke. 1997;28:1043-1048.)
© 1997 American Heart Association, Inc.
Articles |
Chronic Cerebral Hypoperfusion Inhibits Calcium-Induced Long-term Potentiation in Rats
From the Departments of Surgery (L.H.S.S., M.K.M) and Pharmacology (I.S.) and the School of Mathematics and Statistics (N.C.W.), University of Sydney (Australia).
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Background and Purpose Long-term potentiation (LTP) in the rat hippocampus induced by tetanic stimulation is impaired by chronic cerebral hypoperfusion. The effects of chronic cerebral hypoperfusion on other forms of LTP are unknown. Such data could help delineate the pathways of cellular alteration caused by chronic cerebral hypoperfusion. The in vitro phenomenon of calcium-induced LTP was thus examined in rat hippocampal CA1 cells that had undergone chronic hypoperfusion with a reduction in cerebral blood flow of between 25% and 50% maintained for 26 weeks.
Methods Ten Sprague-Dawley rats had a cervical arteriovenous fistula surgically constructed, and an additional 10 animals were used as age-matched controls. Hippocampal slices were prepared after 26 weeks of hypoperfusion, and in vitro extracellular field potential recordings were taken from the Schäffer collateral CA1 region. Properties of LTP induced through transient exposure to a hypercalcemic solution were analyzed.
Results LTP was impaired in animals with an arteriovenous fistula (P<.05). Control animals demonstrated potentiation lasting for the entire 2 hours of recording, whereas fistula animals showed only transient potentiation (<60 minutes) before returning to baseline values.
Conclusions Calcium-induced LTP is impaired by chronic cerebral hypoperfusion. This form of LTP is different from that induced by tetanic stimulation. It is the most sensitive test available for in vitro detection of the changes induced in neuronal function by chronic noninfarctional reductions in cerebral blood flow of 25% to 50% and may indicate that the most basic cellular parameters involving calcium homeostasis and metabolism are being altered. The precise mechanisms remain to be elucidated, and several postulates are discussed.
Key Words: arteriovenous shunt, surgical • calcium • hippocampus • hypoperfusion • potentiation • rats
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Chronic cerebral hypoperfusion in the rat, with reductions in CBF of 25% to 50% maintained for 26 weeks, has been shown to impair in vitro hippocampal LTP1 and in vivo whole animal behavior.2 However, no differences occur at 10 weeks by a similar degree of cerebral hypoperfusion.3 In our previous series of experiments, electrophysiological studies were performed at 10 and 26 weeks after formation of a cervical AVF that induced a state of chronic noninfarctional cerebral hypoperfusion (Fig 1
). In the latter studies, LTP produced by means of
tetanic stimulation was found to be impaired in the AVF
animals.1 The mechanism for this impairment is unclear.
Because no differences were found after 10 weeks of cerebral
hypoperfusion, it was suggested that these changes took time to
manifest.3 Before these studies, only acute
ischemic insults were known to inhibit LTP.4
|
It is known that LTP can be induced by means other than tetanic stimulation, in particular by a transient exposure to an elevated calcium concentration.5 6 7 8 The properties of calcium-induced LTP are somewhat differ- ent from those seen with tetanus-induced LTP.8 9 However, the two phenomena probably share certain common mechanisms. The rationale of using calcium-induced LTP to assess the effects of chronic noninfarctional cerebral hypoperfusion was twofold. First, by using a method other than tetanic stimulation to produce LTP, problems such as achieving the required threshold for activation of neuronal firing and differing degrees of cooperativity among neuronal groups could be overcome. Second, the conclusions reached from our previous studies could be supported or questioned by a different experimental process. It was also hoped that further information could be gleaned concerning possible mechanisms of neuronal impairment induced by chronic noninfarctional cerebral hypoperfusion.
Using a method other than tetanic stimulation to initiate LTP, in this series of experiments we aimed to reexamine our previous conclusions showing that 26 weeks of cerebral hypoperfusion with reductions in CBF of 25% to 50% impaired LTP, as well as to explore the phenomenon of calcium-induced LTP.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
All experimental procedures were approved by our institutional Animal Care and Ethics Committee.
AVF Creation
Ten male Sprague-Dawley rats (weight, 250 to 350 g; age,
8 to 10 weeks) underwent general anesthesia with 1.6%
halothane with the use of a snout mask and spontaneous respiration.
Under direct magnification, a carotid-jugular fistula was formed
between the right internal carotid artery and right external jugular
vein, as detailed by Morgan et al.10 11 12 13 This formed a
functional AVF between the anterior intracranial arterial
circulation and extracranial venous circulation and thus created a
model of cerebral hypoperfusion without an initial ischemic
insult. Morgan et al10 showed that CBF was reduced from a
median of 0.82 to 1.12 mL/g per minute in nonoperated controls to 0.46
to 0.68 mL/g per minute in animals with AVFs using 14C
autoradiography, with no evidence on light microscopy
of infarction at 12 weeks or 26 weeks1 after creation of
the AVF. Ten age-matched rats in our study were used as controls.
After surgery, the rats were allowed to convalesce for 26 weeks in grouped housing, climate-controlled facilities with 12-hour day/night cycles. They were allowed free access to food and water and observed daily for abnormalities of activity or diet.
Hippocampal Slice Preparation
After 26 weeks had elapsed since AVF formation,
electrophysiological studies were performed
on all rats. The animals were anesthetized with halothane and
decapitated. The hippocampus was rapidly removed, and 400-µm slices
were obtained with a McIllwain Tissue Chopper (Mickle Laboratory
Engineering Co Ltd). The slices were placed into a chilled incubation
chamber bubbled with 95% O2/5% CO2 with aCSF
concentration of the following (mmol/L): NaCl 125.3, KCl 3.5,
NaH2PO4 1.25, MgCl2 2,
CaCl2 2, NaHCO3 26, glucose 25 (pH 7.4). Slices
were allowed to recover for at least 1 hour before transfer to a
submerged tissue bath superfused with aCSF (at 8 mL/min at 34±0.5°C)
of the following composition (mmol/L): NaCl 124, KCl 5,
NaH2PO4 1.25, MgCl2 1.3,
CaCl2 2.5, NaHCO3 26, glucose 25 (pH 7.4)
bubbled with 95% O2/5% CO2. Slices were
allowed an additional hour to settle before recordings were
made. Extracellular field potentials were recorded from the CA1
pyramidal layer (recording electrode: glass
micropipette; impedance, 2 to 20 M
; 2 mol/L NaCl) with stimulation
through the Schäffer collaterals (stimulating electrode:
monopolar stainless steel wire with paralyene coating; 125-mm 12°
tapered tungsten tip; impedance, 2 to 5 M). Baseline stimulation
frequency was 0.2 Hz. Control responses were determined with the use of
a stimulus intensity that achieved approximately 30% of the maximal
response and recorded for 30 minutes, with the mean of five
responses recorded every minute. Measurements of the amplitude and
latency of the first population spike as well as the number of spikes
were used as the baseline values for calculating the magnitude of LTP.
The population spike amplitude was measured between peak negativity and
subsequent maximum positivity; latency was measured to peak
negativity.
Calcium-Induced LTP
The regimen described by Reymann et al8 was used to
elicit calcium-induced LTP. To expose the slices to transient
hypercalcemia, the inflow to the tissue chamber was changed to an
alternate solution of aCSF that contained a corresponding reduction in
the NaCl concentration to maintain osmotic equivalence between the two
solutions. This was composed of the following (mmol/L): NaCl 122, KCl
5, NaH2PO4 1.25, MgCl2 2,
CaCl2 4, NaHCO3 26, glucose 25 (pH 7.4). This
solution was preheated to 34°C and also flowed at 8 mL/min, which
effectively exchanged the tissue bath uniformly and completely within
2.5 minutes (which included the time required to fill all the tubing
between the bath and the aCSF solution). The slices were exposed to
this high-calcium solution for 10 minutes, after which the original
aCSF solution was reperfused for the remainder of the experiment.
During this time no stimulation occurred. When perfusion with the
2-mmol/L calcium concentration aCSF solution recommenced, stimulation
was performed only when recordings were actually taken, as
indicated. A single stimulus was given at 7 minutes. At 15, 30, 60, and
120 minutes after the initial exposure to the high-calcium solution,
five stimuli of 5 seconds were given and their means were
recorded.
Statistical Analysis
For each slice, the mean±SD was calculated for the period of
control recording and for the last 30 minutes of
recording after tetanic stimulation. All results were then
pooled. Control recordings between control and AVF slices were
compared by two-sample t tests to detect any differences
before exposure to the high-calcium solution. Two-sample adjusted
t tests were used for samples with unequal
variances.14 The values measured at 7, 15, 30, 60, and 120
minutes were each individually pooled for all control and AVF slice
results. By examination of box and whisker plots for each variable
and on the basis of the method of data collection,
nonparametric testing was considered the appropriate and
most powerful form of statistical comparison. Initially only the
control slice results were examined, with comparisons made among values
during the control period and means recorded at each subsequent
time period, with the use of the Friedman test. Similar comparisons
were made in pooled results from AVF slices. Comparisons were then made
between the mean values obtained at each time period between control
and AVF slices. A Wilcoxon rank sum test (U test)
was used for each pair of comparisons. No corrections for multiple
comparisons were required because various aspects of the same data were
examined rather than the same data being compared repeatedly in
different ways, and as such each variable was discrete. A value of
P<.05 was regarded as significant. Unless specified, all
data are expressed as mean±SD.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The results obtained are summarized in the Table
and Fig 2. There was no statistical difference between
mean values for any variable, before exposure to the hypercalcemic
aCSF solution, between control and AVF slices.
|
|
The responses from a typical control and AVF slice are shown in Fig 2
.
In general, in control slices a mean increase in the population spike
amplitude of 36% from control period values was manifest by t (time)=7
minutes, which increased to 70% at 1 hour and 76% at 2 hours. There
was also a marked and sustained increase in the total number of spikes
and a small reduction in the mean population spike latency, sustained
for the 2 hours of recording. For the AVF slices, a more modest
increase of 33% occurred in the first population spike amplitude at 7
minutes, falling to 24% at 1 hour and 2% at 2 hours. By 120 minutes
all mean values for the three variables had returned to the mean
control period values in the AVF slices. A comparison of control slice
data versus AVF data is shown in the Table. By 60 minutes there were
statistically significant differences between control and AVF data in
terms of the amplitude of the population spike and total number of
spikes (P<.05), which became more pronounced at 120
minutes, with statistically significant differences in the latency of
the population spike and total number of spikes also emerging at that
time. In all cases in which differences emerged, the control slices had
higher mean population spike amplitudes and total numbers of spikes,
with smaller population spike latencies. Aside from a difference in
latency at 15 minutes between the two groups, for the last 60 minutes
of recording there were statistically significant differences
for almost all variables between control and AVF slices.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The results of this series of experiments showed that calcium-induced LTP occurred to a greater extent in control animals than in AVF animals after 26 weeks of a 25% to 50% noninfarctional reduction in CBF. AVF slices demonstrated a lesser degree of calcium-induced LTP and, when compared with the control slices, were unable to sustain the level of potentiation for as long a period of time. These results are of particular interest when compared with the results of experiments on tetanus-induced LTP after 26 weeks of chronic cerebral hypoperfusion.1 These results confirm that chronic noninfarctional changes in CBF of 25% to 50% maintained for 26 weeks impair LTP induced by either tetanic stimulation or transient exposure to a hypercalcemic solution. The magnitudes of induced changes were quite different, however, with tetanus-induced LTP producing more modest relative changes in the measured parameters. This suggests that LTP induced by transient exposure to a hypercalcemic solution differs from tetanic stimulation–induced LTP and that the former may be a more sensitive test for the detection of abnormalities in neuronal function that occur as a result of chronic noninfarctional reductions in CBF of 25% to 50%.
The Role of Calcium
The specific role of calcium in LTP has been established through a
number of cleverly designed studies in the hippocampus. The role of
calcium in LTP was suggested by early experiments in which tetanic
stimulation in the absence of calcium failed to induce
LTP,15 while the lowering of external calcium ion
concentrations reduced the probability of LTP being
initiated.16 The magnitude of LTP was subsequently found
to be highly dependent on the extracellular calcium ion concentration
between 0.8 and 2.0 mmol/L.17 It has been suggested
that the induction of LTP in the CA1 region of the hippocampus involves
a rise in the postsynaptic intracellular calcium ion
concentration.16 18 19 20 Several lines of experimentation
have supported this hypothesis. LTP at this synapse depends on the
direct activation of NMDA receptor–activated calcium
entry.21 22 23 Studies that have directly measured calcium
concentration have demonstrated that NMDA receptor activation leads to
increased calcium concentration in the dendrites of CA1
pyramidal cells in slices.24 Furthermore, LTP
can be blocked by reducing the postsynaptic intracellular calcium
concentration during the induction phase of LTP.20 25 26
Finally, calcium has been shown to play an important role in LTP in the
postsynaptic cell by use of the photolabile calcium ion chelator nitr-5
(which can cause the sudden rise of intracellular calcium
concentrations postsynaptically, leading to a long-lasting potentiation
of synaptic transmission).26 A more recent study in which
the photolabile calcium buffer diazo-4 was used has shown that the rise
in postsynaptic calcium induced by tetanic stimulation lasts a maximum
of 2 to 2.5 seconds, which suggests that increases in the postsynaptic
calcium concentration smaller than required or increases for a duration
shorter than 2 to 2.5 seconds may result in a shorter duration of
potentiation.20
Mechanisms of Impaired LTP in Chronic Cerebral
Hypoperfusion
The present study describes impaired calcium-induced LTP after
26 weeks of mild cerebral hypoperfusion. Calcium-induced LTP is
probably the most sensitive tool available for the assessment of the
subtle effects of mild but chronic reductions in CBF. Although we have
previously shown that infarction of the hippocampus does not occur at
this stage,1 subtle changes are induced in neuronal
structure that ultimately may be responsible for demonstrated changes
in whole animal behavior.2 Given the subtle neuronal
structural alterations that occur, it seems most probable that protein
synthesis and mitochondrial activity are affected. Because protein
kinases are required for the conversion of tetanus-induced short-term
potentiation into LTP,27 it seems probable that protein
synthesis impairment is the likely culprit for the demonstrated
changes. Such impairment has already been demonstrated for slightly
more marked reductions in CBF,28 29 and it seems likely
that such a process would occur during more chronic insults.
Conclusions
The results from these experiments provide further support for the
assertion that chronic noninfarctional reductions in CBF maintained for
26 weeks impair neuronal function, confirming our previous findings.
Further information is provided regarding hypercalcemia-induced LTP,
and in particular we showed that this phenomenon is probably the most
sensitive test available for detecting the
electrophysiological effects of subtle yet
chronic reductions in CBF. It is suggested that hypercalcemia-induced
LTP is different from tetanus-induced LTP, as shown by the magnitude of
our responses, although the general patterns were similar in this
context in both control and AVF animals. Hypercalcemia-induced LTP may
show changes with even more modest reductions in CBF.
Chronic cerebral hypoperfusion of this magnitude affects neuronal structure and whole animal function,2 and this set of experiments, in addition to more clearly demonstrating the differences between normal and hypoperfused animals, suggests that some of the most basic processes in neurons in terms of calcium metabolism are altered. LTP is a calcium-mediated event, yet many other cellular functions, including mitochondrial respiration, rely on calcium for efficient cellular function. With this in mind, apart from highlighting a more sensitive investigative tool, these experiments suggest that some of the most basic neuronal functions involving calcium homeostasis may be altered, which could partly explain the histopathological and behavioral findings we have described. Mitochondria require calcium, and mitochondria play a role in protein synthesis, a most basic cellular function that affects all functions of the neuron. It seems intuitively likely that this degree of ischemia probably acts by an inhibition of cellular protein synthesis. This remains to be demonstrated, and the precise mechanisms still remain to be identified, although it is now clear that chronically maintained yet mild reductions in CBF affect some of the most basic properties of neuronal function in terms of calcium regulation.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This research is part of an ongoing project investigating the pathophysiology of chronic hypoperfusion and cerebral arteriovenous malformations. This study was supported in part by the following: National Health and Medical Research Council, Clive and Vera Ramaciotti Research Foundation, Royal Australasian College of Surgeons, and the Australian Brain Foundation.
|
Footnotes |
|---|
Reprint requests to M.K. Morgan, MD, FRACS, Department of Neurosurgery, Level 7, Royal North Shore Hospital, St Leonards, NSW 2065, Australia.
Received September 4, 1996; revision received January 10, 1997; accepted February 3, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Sekhon LHS, Morgan MK, Spence I, Weber NC. Chronic cerebral hypoperfusion and impaired neuronal function in rats. Stroke. 1994;25:1022-1027.[Abstract]
2. Sekhon LHS, Morgan MK, Spence I, Weber NC. Chronic cerebral hypoperfusion: pathological and behavioral consequences. Neurosurgery. 1997;40:548-556.[Medline] [Order article via Infotrieve]
3. Sekhon LHS, Spence I, Morgan MK, Weber NC. Chronic cerebral hypoperfusion in the rat: temporal delineation of effects and the in vitro ischemic threshold. Brain Res. 1995;704:107-111.[Medline] [Order article via Infotrieve]
4. Kirino T, Robinson HPC, Miwa A, Tamura A, Kawai N. Disturbance of membrane function preceding ischemic delayed neuronal death in the gerbil hippocampus. J Cereb Blood Flow Metab. 1992;12:408-417.[Medline] [Order article via Infotrieve]
5. Turner RW, Baimbridge KG, Miller JJ. Calcium-induced long-term potentiation in the hippocampus. Neuroscience. 1982;7:1411-1416.[Medline] [Order article via Infotrieve]
6. Bliss TVP, Dolphin AC, Feasey KJ. Elevated calcium induces a long-lasting potentiation of commissural responses in hippocampal CA3 cells of the rat in vivo. J Physiol (Lond). 1984;350:65P. Abstract.
7. Bliss TVP, Errington ML, Lynch MA. Calcium-induced long-term potentiation in the dentate gyrus is accompanied by a sustained increase in glutamate release. In: Hicks TP, Lodge D, eds. Excitatory Amino Acid Transmitters. New York, NY: Alan R Liss, Inc; 1986:337-340.
8. Reymann KG, Matthies HK, Frey U, Vorobyev VS, Matthies H. Calcium-induced long-term potentiation in the hippocampal slice: characterization of the time course and conditions. Brain Res Bull. 1986;17:291-296.[Medline] [Order article via Infotrieve]
9. Melcher BPC, Pennartz CMA, Lopes da Silva FH. Differential effects of elevated extracellular calcium concentrations on field potentials in dentate gyrus and CA1 of the rat hippocampal slice preparation. Neurosci Lett. 1987;77:37-42.[Medline] [Order article via Infotrieve]
10. Morgan MK, Anderson RE, Sundt TM Jr. A model of the pathophysiology of cerebral arteriovenous malformations by a carotid-jugular fistula in the rat. Brain Res. 1989;496:241-250.[Medline] [Order article via Infotrieve]
11. Morgan MK, Anderson RE, Sundt TM Jr. The effects of hyperventilation on cerebral blood flow in the rat with an open and closed carotid-jugular fistula. Neurosurgery. 1989;25:606-612.[Medline] [Order article via Infotrieve]
12. Morgan MK, Johnston I, Besser M, Baines D. Cerebral arteriovenous malformations, steal, and the hypertensive breakthrough threshold: an experimental study in rats. J Neurosurg. 1987;66:563-567.[Medline] [Order article via Infotrieve]
13. Morgan MK, Sundt TM Jr, Anderson RE. The haemodynamic consequences of a carotid-jugular fistula in the rat during hypocapnoea. J Clin Neurosci.. 1994;1:193-196.[Medline] [Order article via Infotrieve]
14. Scheffe H. Practical solutions of the Behrens-Fisher problem. J Am Stat Assoc. 1970;65:1501-1508.
15. Wigstrom H, Swann JW, Andersen P. Calcium dependency of synaptic long-lasting potentiation in the hippocampal slice. Acta Physiol Scand. 1979;105:126-128.[Medline] [Order article via Infotrieve]
16. Dunwiddie TV, Lynch G. The relationship between extracellular calcium concentration and the induction of hippocampal long-term potentiation. Brain Res. 1979;169:103-110.[Medline] [Order article via Infotrieve]
17. Mulkeen D, Anwyl R, Rowan M. The effects of external calcium on long-term potentiation in the rat hippocampal slice. Brain Res. 1988;447:234-238.[Medline] [Order article via Infotrieve]
18. Eccles JC. Calcium in long-term potentiation as a model for memory. Neuroscience. 1983;10:1071-1081.[Medline] [Order article via Infotrieve]
19. Smith SJ. Progress on LTP at hippocampal synapses: a postsynaptic Ca2+ trigger for memory storage? Trends Neurosci. 1987;10:142-144.
20. Malenka RC, Lancaster B, Zucker RS. Temporal limits on the rise in postsynaptic calcium required for the induction of long-term potentiation. Neuron. 1992;9:121-128.[Medline] [Order article via Infotrieve]
21. Jahr CE, Stevens CF. Glutamate activates multiple single channel conductances in hippocampal neurons. Nature. 1987;325:522-525.[Medline] [Order article via Infotrieve]
22.
Mayer ML, Westbrook GL. Permeation and block of
N-methyl-D-aspartic acid receptor channels by divalent cations in mouse
central neurons. J Physiol (Lond). 1987;394:501-528.
23. Ascher P, Nowak L. The role of divalent cations in the N-methyl-D-aspartate responses of mouse central neurones in culture. J Physiol (Lond). 1988,399:247-266.
24. Regehr WG, Tank DW. Postsynaptic NMDA receptor-mediated calcium accumulation in hippocampal CA1 pyramidal cell dendrites. Nature. 1990;345:807-810.[Medline] [Order article via Infotrieve]
25. Lynch G, Larson J, Kelso S, Barrionuevo G, Schottler F. Intracellular injections of EGTA block induction of hippocampal long-term potentiation. Nature. 1983;305:719-721.[Medline] [Order article via Infotrieve]
26.
Malenka RC, Kauer JA, Zucker RS, Nicoll RA.
Postsynaptic calcium is sufficient for potentiation of hippocampal
synaptic transmission. Science. 1988;242:81-84.
27. Bliss TVP, Collingridge GL. A synaptic model of memory: long-term potentiation in the hippocampus. Nature. 1993;361:31-39.[Medline] [Order article via Infotrieve]
28. Mies G, Ishimaru S, Xie Y, Hossmann K-A. Thresholds of protein synthesis and energy metabolism in focal brain ischemia. In: Krieglstein J, Oberpichler H, eds. Pharmacology of Cerebral Ischemia 1990. Stuttgart, Germany: Wissenschaftliche Verlagsgesellschaft mbH; 1990:89-95.
29. Mies G, Ishimaru S, Xie Y, Seo K, Hossmann K-A. Ischemic thresholds of cerebral protein synthesis and energy state following middle cerebral artery occlusion in rat. J Cereb Blood Flow Metab. 1991;11:753-761.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  P. M. Klinge, A. Samii, A. Muhlendyck, K. Visnyei, G.-J. Meyer, G. F. Walter, G. D. Silverberg, and T. Brinker Cerebral Hypoperfusion and Delayed Hippocampal Response After Induction of Adult Kaolin Hydrocephalus Stroke, January 1, 2003; 34(1): 193 - 199. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||