long-term potentiation trains induce mossy fiber sprouting
TRANSCRIPT
Ž .Brain Research 775 1997 193–197
Short communication
Long-term potentiation trains induce mossy fiber sprouting
Beth Adams a, Melvin Lee a, Margaret Fahnestock b, Ronald J. Racine a,)
a Department of Psychology, McMaster UniÕersity, Hamilton, Ont. L8S 4K1, Canadab Department of Biomedical Sciences, McMaster UniÕersity, Hamilton, Ont. L8S 4K1, Canada
Accepted 19 August 1997
Abstract
It has been shown that both amygdaloid and hippocampal kindling induce sprouting of the mossy fibers in the dentate gyrus. In thisŽ .study, we investigated whether non-epileptogenic stimulation could also induce mossy fiber sprouting. Long-term potentiation LTP was
induced in the dentate gyrus by the application of brief, high-frequency trains to the perforant path. The potentiating stimulation wasapplied each day for 10 days, and the tissue was prepared for Timm labelling 7 days later. Sprouting was significantly increased in theLTP group compared to the implanted control rats. These results suggest that mossy fiber sprouting is not damage-induced and isdependent on neuronal activation. q 1997 Elsevier Science B.V.
Keywords: Long-term potentiation; Kindling; Hippocampus; Mossy fiber; Sprouting; Plasticity
Kindling is an experimental epilepsy model which canbe defined as the progressive development of electroen-cephalographic and behavioural seizure activity produced
w xby spaced and repeated epileptogenic stimulation 9,11 .Recent research has shown that kindling induces a reorga-nization of synaptic connections of the mossy fiber path-
w xway 13 . To date, however, neither the necessary condi-tions for, nor the functional significance of, this kindling-induced structural reorganization is clear. One hypothesisis that kindling produces neuronal loss in the hippocampusand that the mossy fibers may sprout to replace synapses
w xvacated by degenerating axons 5 . Furthermore, it hasbeen suggested that these sprouting mossy fibers maycontribute to enhanced excitability and increased suscepti-bility to seizures. In this case, hippocampal neuronal lossmay be both a cause and an effect of seizures. On the otherhand, kindling-induced mossy fiber sprouting has alsobeen reported in the absence of neuronal loss or degenera-
w xtion 12 . One explanation for these findings is that thedegenerative effects were too small to be easily measured.Another possibility is that mossy fiber sprouting is induced
w xby neuronal activation itself 6 . Whether triggered by
) Ž .Corresponding author. Fax: q1 905 529-6225; E-mail: [email protected]
neuronal loss or activation, there is evidence that neuronalsprouting may be regulated by a variety of growth factorsw x7 .
We have recently demonstrated that intraventricularŽ .nerve growth factor NGF infusions increase kindling-in-
duced mossy fiber sprouting in the absence of hilar neu-w xronal loss 1 . These findings appear to exclude the possi-
bility that the mossy fiber sprouting is triggered by anyreadily detectable neuron losses. These findings also raisethe possibility that kindling-induced mossy fiber sprouting
w xmay be dependent, as in the case in the periphery 7 , onthe co-involvement of neuronal activation and growth fac-tors. The purpose of this study was to investigate whether
Žnon-epileptogenic neuronal activation i.e., stimulation thatdoes not evoke an epileptiform after-discharge and pre-
.sumably does not produce degenerative effects can inducemossy fiber sprouting. One example of non-epileptogenicstimulation is the brief high-frequency stimulus trains used
Ž .to induce long-term potentiation LTP . LTP refers to anincrease in synaptic strength produced by high-frequencystimulation of excitatory afferents and can be defined as astable, long-lasting increase in the amplitude of post-syn-aptic responses evoked in a neuronal pathway following
w xactivation of that pathway via brief tetanic stimulation 3 .LTP is considered to be a synaptic model for memory andis the leading candidate mechanism for an informationstorage mechanism in the mammalian central nervous sys-
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 97 01061-5
( )B. Adams et al.rBrain Research 775 1997 193–197194
w xtem 2 . To date, there have been no reports of LTP-in-duced mossy fiber sprouting or LTP-induced neuronaldegeneration.
Sixteen male Long–Evans rats weighing between 300and 400 g were used. Rats were anaesthetized with sodium
Ž .pentobarbitol 65 mgrkg and implanted with a bipolarelectrode made from teflon-coated, stainless steel wiresŽ .diameter: 120 mm in the right perforant path. Stereotaxic
w xcoordinates 10 were 7.6 mm posterior and 4.1 mm lateralto bregma and 3.3 mm below the skull surface. Theelectrode was held in place by dental acrylic and threestainless steel screws inserted into the skull. Following a2-week recovery period, rats were randomly assigned to
Ž .either an LTP condition ns8 or a control conditionŽ .ns8 . Implanted control rats remained in the colony for32 days post-surgery. We did not implant electrodes in thedentate gyrus to monitor the progression of potentiationbecause it would have substantially interfered with subse-quent histological analyses and because the stimulationprotocol that was used reliably triggers LTP in our labora-tory.
To ensure that epileptogenic activity was absent fromrats prior to LTP stimulations, a switching circuit was usedto record field activity from the perforant path electrodeduring the first session of high-frequency stimulation. Ratsin the LTP group were given one stimulation session a dayfor 11 consecutive days. During each session, 30 eight-pulse trains were delivered to the perforant path at a rate ofone train every 10 s. Trains were delivered at a frequencyof 400 Hz and a pulse intensity of 1000 mA. During thelast session of LTP stimulations, post-train electrical activ-ity was recorded again to ensure the continued absence ofepileptogenic activity. Control rats were match-handled.Rats remained in the colony for 7 days following thedelivery of LTP trains to allow maximal levels of mossyfiber sprouting.
On Day 32 post-surgery, rats were anaesthetized withsodium pentobarbitol and rats were perfused with 50 ml of
Ža sodium sulfide solution 8.9 g Na SP9H O, 10.9 g2 2.sucrose, 1.19 g Na PO PH O per 100 ml dH O at room2 4 2 2
temperature. Following perfusion, brains were frozen inisopentane cooled to y408C on dry ice and stored aty708C. Horizontal 40 mm sections of the hippocampalarea at 4.1–8.1 mm ventral to bregma were sectionedusing a cryostat at y188C and sections were mounted onchromium potassium sulfate-coated slides.
The Timm method stains neural elements containingŽ 2qheavy metals i.e., the high Zn content of the terminals
.of the mossy fibers . Brain sections were processed using aw xmodified Timm method 14 for the analysis of mossy
fiber sprouting. To minimize variability in Timm stainingbetween groups, tissue sections from the experimental andcontrol groups were processed simultaneously. To ensureobjectivity in the data analysis, slides were coded and allsubsequent analyses were done by an observer who wasunaware of the treatment of the animal.
Brain sections were examined at 50= magnification bycreating a digitized image using an MCID image analyses
Žsystem Micro Computer Imaging Device, Brock Univer-.sity, St. Catherine’s Ontario, Canada attached to a light
Ž .microscope Zeiss Axioskop with a high-resolution chargeŽ .coupled device camera MTI CCD 72 . Timm densitom-
w xetry was conducted as described by Adams et al. 1 .Briefly, the density of Timm granules in the hippocampalCA3 region was measured by placing an open circle cursorŽ 2 .0.013 cm at eight adjacent positions along the stratumoriens of the CA3. Background values were provided byreadings at eight cursor placements in the stratum radiatumof the CA3. The density of Timm granules in the innermolecular layer was measured at nine adjacent cursorpositions by placing one cursor position above the genu ofthe hilus and four cursor positions to the right and the leftof this cursor. To control for variations in backgroundTimm staining density from section to section, the densityreadings in the stratum oriens and in the IML were dividedby the background density values from the stratum radia-tum for each section. This provided a ratio between thestratum oriens or the IML density value per section forboth groups. Density measurements were evaluated from
Ž .10 brain sections per rat 400 mm apart for both the rightand left sides of the brain.
w Ž .x w ŽA 4-way ANOVA 2= 10=2=8 Group LTP or. Ž Ž .control = Section Depth ventral to dorsal = Brain
Ž . .xHemisphere left and right =Cursor Position was con-ducted on the Timm granule density ratios obtained fromthe CA3 region. There was a significant Group=Section
ŽDepth=Cursor Position interaction F s1.89; P-63, 819.0.001 showing that Timm granule density was increased
in the LTP group at cursor positions that are closer to thedentate gyrus and in more dorsal brain sections compared
Ž .to the implanted controls Fig. 1 .w Ž .x w ŽA 2= 10=2=9 Group= Section Depth=Brain
.xHemisphere=Cursor Position ANOVA was conductedon the Timm granule densities obtained from the IMLregion. There was a significant Group=Section Depth=
Ž . .Cursor Position interaction F s1.65, P-0.00172, 936
showing that Timm granule density was greatest in theLTP group in the genu region of the dentate gyrus and in
Žmore ventral sections compared to the control group Fig..2 .
These findings provide the first evidence that LTPtrains induce mossy fiber sprouting in the hippocampalCA3 and IML regions. Moreover, these findings suggestthat mossy fiber sprouting can occur in the absence ofneuronal degeneration. Rather, these results support thehypothesis that mossy fiber sprouting is dependent onneuronal activation.
It has been reported that seizure-induced changes inhippocampal neurotrophic factor mRNA and protein ex-
w xpression are correlated with synaptic reorganization 8 .This raises the possibility that neurotrophic factors mayplay an important role in activity-dependent plasticity in
( )B. Adams et al.rBrain Research 775 1997 193–197 195
epileptogenesis. Similarly, recent research has shown en-hanced neurotrophin and neurotrophin receptor mRNAexpression specific to LTP in the hippocampus of freely
w xmoving rats 4 . These results provide additional evidencefor a physiological role for neurotrophic factor and neu-rotrophic factor receptor regulation in activity-dependent
Ž .Fig. 1. Photomicrographs of Timm staining in the CA3 region. Representative examples of area CA3 of an implanted control rat A and a rat that receivedŽ .LTP trains B . Arrows point to Timm granules in the stratum oriens of the CA3. Note that Timm granule density in the CA3 region is increased in the
LTP condition compared to the control condition.
( )B. Adams et al.rBrain Research 775 1997 193–197196
Ž .Fig. 2. Photomicrographs of Timm staining in the IML. Representative examples of IML region of an implanted control rat A and a rat that received LTPtrains. Arrows point to the band of Timm staining in the IML region. Note that the density of the band of Timm staining in the IML region is greater in theLTP condition compared to the control condition.
synaptic plasticity in the adult hippocampus. Moreover,these findings suggest that an LTP-induced up-regulationof growth factors may underlie the LTP-induced mossy
fiber sprouting observed in the present study. Additionalresearch is required to investigate the functional signifi-cance of activation-induced mossy fiber sprouting.
( )B. Adams et al.rBrain Research 775 1997 193–197 197
1. Note added in proof
We came across the following reference while proofingthe galleys: M.L. Escobar, E.J. Barea-Rodriguez, B.E.Derrick, J.A. Reyes, J.L. Martinez Jr., Opioid receptormodulation of mossy fiber synaptogenesis: independence
Ž .from long-term potentiation, Brain Res. 751 1997 330–335. In this paper, they show that sprouting can be inducedin the mossy fiber pathway by direct, non-epileptogenicstimulation of that pathway.
Acknowledgements
This work was supported by grants from the Neuro-Ž . Žscience Networks Centres of Excellence NCE R.J.R. and
. Ž .M.F. , the Medical Research Council of Canada MRCŽ .R.J.R. and M.F. the Natural Sciences and Engineering
Ž . Ž .Research Council of Canada NSERC R.J.R. . B.A. wassupported by a post-graduate scholarship from NSERCŽ .PGS B , a supplement from the NCE, and a studentshipfrom the Savoy Foundation.
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