morphogenetic effect of kainate on adult hippocampal neurons associated with a prolonged expression...

~ Pergamon 0306-4522(94)00463-3

Neuroscience Vol. 64, No. 3, pp. 665-674, 1995 Elsevier Science Ltd

Copyright L7 1995 IBRO Printed in Great Britain. All rights reserved

0306-4522/95 $9.50 + 0.00

MORPHOGENETIC EFFECT OF KAINATE ON ADULT HIPPOCAMPAL NEURONS ASSOCIATED WITH A PROLONGED EXPRESSION OF BRAIN-DERIVED

NEUROTROPHIC FACTOR

F. SUZUKI,* M.-P. JUNIER,* D. GUILHEM,* J.-C. SO'RENSENt and B. ONTENIENTE*~

*Institut National de la Sant6 et de la Recherche M6dicale CJF 91-02, 8 rue du G6n6ral Sarrail, F-94010 Cr6teil cedex, France

tPharmabiotec, Department of Anatomy, Odense University, DK-5000 Odense C., Denmark

Abstraet--lntraperitoneal or intrahippocampal injections of kainate induce both hippocampal cell death and axonal remodeling of the dentate gyrus granular neurons. We report here that injection of kainate into the dorsal hippocampus of adult mice may also trigger a conspicuous and long-lasting global trophic response of granule cells. Morphological changes include somatic and dendritic growth and increased nuclear volume with ultrastructaral features characteristic of neuronal development. The trophic response is correlated with a specific overexpression of brain-derived neurotrophic factor that is maintained for at least six months.

This shows that plasticity in adult neurons can, in addition to axonal remodeling, extend to generalized cell growth. Our results further suggest that brain-derived neurotrophic factor could be involved in the activation and/or maintenance of this phenomenon.

Neuronal plasticity in the adult mammalian brain has been mainly investigated with respect to chemical or structural remodeling of axonal or dendritic patterns. The hippocampus is a privileged site for the study of brain plasticity: (i) long-term potentiation, a major example of molecular and morphological long-lasting modification of synaptic efficiency, has been evi- denced there (see Ref. 9 for review); (ii) collateral axonal sprouting of mossy fibers, axons of the dentate gyrus (DG) granular neurons, can be activated by increased neuronal activity, loss of target cells or partial denervat ion; 7~14"2t'34"43"51'56 (iii) collateral axonal sprouting of hippocampal afferent systems follows partial denervation; 54 (iv) the hippocampus contains the highest brain levels of neurotrophic factors, that are major determinants of neuronal plasticity (see below).

Hippocampal neurons are particularly sensitive to the neurotoxic action of the glutamate analog, kainate (KA). Seizure activity induced by systemic administration of KA is associated with neuronal loss in CA3 and subsequent permanent changes in the axonal architecture of dentate granule cells. 7'43 Collat- eral sprouting and synaptic reorganization of mossy fibers is also observed after intraparenchymal administration of KA that involves neuronal loss in CAI

~+To whom correspondence should be addressed. Abbreviations: BDNF, brain-derived neurotrophic factor;

DG, dentate gyrus; KA, kainate; NGF, nerve growth factor.

and increases neuronal activity in the gyrus. 6'13'44 In addition to its excitotoxic properties, KA may elicit trophic responses in specific neuronal populations. 46

Neuronal activation in the hippocampus is consis- tantly correlated with modification of the expression of neurotrophic factors. Nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin-3, together with neurotrophin 4-5, consti- tute the neurotrophin f a m i l y Y 7'27"35"41 The highest brain levels of NGF, BDNF and neurotrophin-3 are found in the hippocampus, with distinct expression patternsJ 8'26'47 While neurons expressing NGF mRNA are scattered in Ammon's horn and the hilar region, 1,~8'23 BDNF mRNA is more abundant within CA2 and CA3 pyramidal neurons and in the O G . 18"26"47'55 Increases in B D N F or N G F genes expression have been described in hippocampal neurons after depolarization in vitro, 57 after stimulation of non-NMDA 57 and NMDA receptors, 25'2s after chemically -2'24'57 or electrically -~9'3°'4~ induced seizures and following stimulation of afferent systems. 2°'36 On the other hand, neurotrophin-3 mRNA expression is unchanged or decreased in hippocampal neurons after hippocampal lesion, z'49

On the basis of recent evidence that neurotrophic factors can stimulate axonal sprouting in the brain, (see Ref. 4 for review) their implication in KA- induced axonal plasticity in the DG has been suggested) 3'5° However, variations in the expression of neurotrophins mRNA induced by the experimental paradigms mentioned above are transient, peaking

665

666 F. Suzuki et al.

between 1 and 6 h following stimulation before resuming to normal levels after 24-48 h. 22 However, evidence for a more prolonged, NGF-l ike trophic activity in extracts of hippocampal tissue after KA- induced seizures has recently been given. 38 Such a long-lasting expression would be more consistent with a causal relationship between an increase in neurotrophic factors and morphological remodeling associated with epileptic syndromes.

In order to investigate further the implication of neurotrophins in hippocampal neuronal plasticity, we have established a paradigm that leads to a more prolonged expression of neurotrophin genes. Re- stricted injections of various doses of K A were performed into the mouse dorsal hippocampus.

EXPERIMENTAL PROCEDURES

Surgical procedures and preparation of tissue samples

Adult male mice (C57B1/6 × DBA, 30 35g, Charles River) were anesthetized with chloral hydrate (350 mg/kg, i.p.) and placed into a stereotaxic frame. They received a unilateral injection of KA (concentrations ranging from 2 to 0.5 nM, pH 7.4) in 0.05/~1 of saline into the left dorsal hippocampus (coordinates from Bregma: A: - 2 ; L: 1.8; D: 2 mm from dura). Some animals were injected with smaller (0.02 #1) volumes of the same KA solution either more medially (L: 1.5) or more laterally (L: 2). Control animals were injected with the same amount of saline. Animals were killed 1 day to 16 weeks after surgery by either intra-aortic perfusion of 4% paraformaldehyde~0.5% glutaraldehyde in 0.1 M phosphate-buffered saline (histology) or decapitation (in situ hybridization). Perfused brains were post-fixed overnight in 4% paraformaldehyde, cryoprotected in 30% sucrose for 36 h and cut with a cryostat (20/~m). Sections were mounted on glass slides, dehydrated and stained with Cresyl Violet.

Electron microscopy'

Four animals were perfused at one, two, four and eight weeks after injection, respectively, with 5% glutaraldehyde in 0.1 M phosphate buffer. Fifty-micrometer sections were cut on a Vibratome, post-fixed in 0.2% osmium tetroxide, dehydrated in gradded ethanol solutions and embedded in Epon. Ultrathin sections were cut with a LKB UM5 ultra- microtome and observed after counterstaining with uranyl acetate and lead citrate on a Phillips M10 electron micro- scope.

In situ hybridization

Three independent series were performed. Each series contained slides from two different animals at each post-injection date (6 h, 24 h, two days, four days, eight days, two weeks, four weeks, eight weeks, 16 weeks and 23 weeks). Fresh frozen tissue sections (20 #m) through the hippocampus were cut on a cryostat and thawed on to 3- aminopropyl-triethoxysilane(Fluka)-coated slides. After fixation for 10min in 4% paraformaldehyde, the sections were processed for isotopic in situ hybridization as described t° with 33P-labelled RNA probes. The sections were prehybridized at 42°C and hybridized for 16 h at 42°C with 33P-labelled BDNF, NGF, neurotrophin-3 or trkB anti- sense or sense riboprobe (107 c.p.m./ml). BDNF probes were transcribed from a pGEM7Z vector containing a 372bp sequence of the rat cDNA (nt 369-74 141). NGF probes were transcribed from the mouse cDNA (nt 307-50756). Neuro- trophin-3 probe were transcribed from the mouse cDNA (nt 658-8674~). TrkB probes were transcribed from a pBlue- script vector containing a 433 bp sequence of the rat cDNA (nt 2151 2584) specific of the full-length TrkB. 5° Plasmids

were linearized and DNA sequences were transcribed by using T7 (NGF and TrkB sense probes, BDNF and NT-3 anti-sense probes), T3 (NGF and trkB anti-sense probes) or SP6 (BDNF sense probe) RNA polymerase in the presence of 33P-CTP (Amersham, specific activity > 110 Tbq/mmol).

After hybridization, the sections were treated with RNase A (20pg/ml), washed four times in 0.2 x saline sodium phosphate (SSPE) and air-dried before exposition to fl-max Hyperfilm (Amersham) for two days. No specific labeling was observed with sense probes.

The levels of mRNAs were quantified by computerized image analysis (Samba/2005, TITN, Alcatel, France). In each series, changes in optical densities were estimated on the KA-injected side as a percentage of the contralateral side. To avoid underestimations due to the progressive enlargement of the granular layer, values were measured on the total area of the layer outlined on adjacent Cresyl Violet-stained sections. Optical densities of pixels overlying regions of interest from three sections per mouse were corrected for background and averaged. Values obtained for each experimental point in the three series were pooled to calculate the mean value and S.E.M.

RESULTS

Behavioral observations

Characteristic seizure activity was observed in animals that had received intrahippocampal injections of 2 nM of KA. No behavioral signs of seizure activity and no chronic, recurrent, seizures were observed in animals injected with concentrations of K A lower than 1.5nM. One nanomolar induced a transient (45 min to 1 h) apathy and limited motor activity. Lower concentrations had no behavioral effect.

Morphogenetic response of granule cells

The injection of 2 nM K A into the dorsal hippocampus of adult mice induced the complete degeneration of Ammon ' s horn and DG. The injection of concentrations ranging from 0.8 to 1.5 nM K A induced a typical pattern of local damage, with destruction of pyramidal and non-pyramidal cells in the CA 1 field and occasionally a partial destruction of CA3. These effects occurred over a period of 48h. Although varying with the dose and location of the injection site, the neuronal degeneration within Ammon ' s horn progressively extended within weeks to CA4, CA2 and finally CA3. The injection of concentrations lower than 0.8 nM resulted in a very discrete neuronal loss in CA1, with no apparent modifications of other hippocampal structures.

In addition, concentrations of K A of 1.5 and 1 nM induced remarkable morphological changes in the granular layer of the DG. A progressive enlargement of the layer was seen from four days up to eight weeks (Figs 1, 2). The increase was dramatic (up to 240%) within the first two weeks and then progressed more slowly (Fig. 2). At eight weeks, the D G had completely filled the space left by the atrophic Ammon ' s horn. Neuronal hypertrophy was maintained up to 23 weeks, the longest survival time examined.

Higher magnification of Cresyl Violet-stained sections showed that the enlargement of the granule cell layer resulted from a concomitant increase in neur-

Morphogenetic effect of kainate in adult hippocampus 667

Fig. 1. Hypertrophy of the mouse dentate gyrus granule cell layer after injection of kainate (KA) in the dorsal hippocampus (star) of adult mouse. Cresyl Violet staining of the hippocampal formation of a normal mouse (A) and after treatment with 1 nM KA (B-F). The granular layer of the dentate gyrus (DG) progressively increases in width. Note the disappearance of Ammon's horn (AH) except for remaining CA3 pyramidal neurons (arrows in C and D). (B) Seven days, (C) two weeks, (D) eight weeks, (E) 16

weeks and (F) 23 weeks after injection. Scale bar = 1 mm.

onal size and intercellular space (Fig. 3). The neuronal hypertrophy involved proximal dendrites, soma and nucleus (Figs 3, 4). After four weeks the somatic and nuclear size in hypertrophied neurons was four- fold that of normal granular neurons. The absolute number of granule cells seemed unchanged on Cresyl Violet-stained sections. Complex axonal modifications also occurred, including sprouting of uniden- tified axons, that will be described in detail elsewhere.

Electron microscopy revealed a dissociation of nuclear chromatin aggregates (Fig. 4A, B) and a development of endoplasmic reticulum and number of polyribosomes (Fig. 4C). No degenerating neurons

and no signs of mitotic activity were observed with electron microscopy in the granular layer, consistent with the lack of evident modification of the cell number shown by Cresyl Violet stainings.

Morphogenetie effects are not related to the loss of CA 3 target neurons

To assess for a direct relation between KA and the morphological effects, focal injections of smaller volumes of KA were performed in different areas of the hippocampus. Lateral injections of 0.02 ~1 of 1 nM KA at the level of CA2 (Fig. 5A) induced a selective destruction of CA3 while partially preserving CA 1. In


450 -

400

350,

300 -

250 -

200-

150

100

50 0 20 40 8o t20

Days post-injection

Fig. 2. Quantitation of the thickness of the granule cell layer after injection of 1 nM KA into the dorsal hippocampus of adult mice. Values (mean + S.E.M.) are given as a percentage of the normal side. n = 4-6 animals per group for each point.

such cases, only the most lateral neurons of the gyrus, situated close to the injection site, were hypertrophied. Medial injections, at the level of the subicu- lum, induced a partial degeneration of CAI while preserving completely CA2, CA3 and CA4 neurons. Despite the total sparing of CA3, such injections induced a general hypertrophy of granular neurons, with the exception of the most lateral neurons (Fig. 5B).

Expression o f neurotrophins

Changes in neurotrophin gene expression were estimated by the levels of their respective m R N A observed by in situ hybridization. As repeatedly reported, z'z4,25,28'57 an increase in the expression of NGF, BDNF and a slight decrease in neurotrophin-3

Fig. 3. Light microscopic aspect of the granule cell layer eight weeks after intrahippocampal injection of KA. Staining with Cresyl Violet reveals an increased spacing of granule cells, enlargement of the nucleus and cytoplasm and thickening of proximal dendrites (arrow) in KA-treated (B) compared to normal (A)

animals. Scale bar = 20/1 m.

Fig. 4. Electron micrograph of granule cells in normal (A) and KA-treated (B, C) animals four weeks after KA administration. Aggregates of nuclear chromatin (arrows) have disappeared in granular neurons of KA-treated animals which show an abundance of rough endoplasmic reticulum with increased number

of polyribosomes, n, nucleolus. Scale bar = 2.3 #m,


~i ̧ ~i~.il ~ ~i~i,!.~ ~ ~

Fig. 5. Effects of varying placements of KA injection sites in the dorsal hippocampus. (A) Injections into the lateral part of the hippocampus (star) induce the degeneration of CA3 pyramidal neurons without affecting granule cells except for cells adjacent to the injection site (arrow). (B) Injections into the medial part of the hippocampus (star) preserve CA3 from degeneration and induce granule cells hypertrophy

(arrow). Two weeks after KA administration.

mRNA were observed in DG cells within 3-6 h following the injection of KA (not shown). However, after this first peak, BDNF mRNA increased again from 24 h post-injection to reach a maximal level at seven days (270% of normal levels, Figs 6, 7A). BDNF mRNA levels dropped to 145% during the third and fourth weeks post-injection and slowly increased again afterwards. Sixteen weeks (four months) post-injection, BDNF mRNA levels were still at 160% of normal levels. High levels of BDNF mRNA were maintained up to 23 weeks (Fig. 6).

In contrast to BDNF, N G F and neurotrophin-3 mRNAs in granule cells had essentially returned to basal levels after a few hours, except for a very slight decrease in neurotrophin-3 that was maintained for two months (Fig. 7A).

Expression of Trks

In contrast with recent observations of a correlated overexpression of BDNF and its high-affinity receptor, trkB, mRNA levels of trkB were not significantly changed in granule cells-during the whole period of investigation (Figs 6E, F; 7B). No changes of trkA and trkC, the respective high affinity receptors for N G F and neurotrophin-3, were observed at any time.

D I S C U S S I O N

Our results show that a single injection of KA into the adult mouse brain can set in motion processes that, directly or indirectly, lead to cell growth. The histological and ultrastructural characteristics of the morphogenetic effects observed in this study resemble those observed during the development of hippocampal structures 5'j2 and strongly suggest that this phenomenon corresponds to the return to a developmental program. Electron microscopy revealed a dissociation of nuclear chromatin aggregates which

indicates increased transcriptional activity, and a development of endoplasmic reticulum and number of polyribosomes which indicates an increase of protein synthesis. A t r o p h i c effect of KA has been described on a A-type horizontal cells of the retina, 46 including atypical sprouting of processes into adjacent layers. Although not indicated by the authors, an increase in somatic size was also apparent on the illustrations. The temporal correlation of the morphological modifications observed in our paradigm with a specific and sustained overexpression of BDNF suggests that they are triggered and/or maintained by BDNF. However, in contrast with previous de- scriptions of correlated increases of BDNF and its high affinity receptor, 2'n no long-lasting modifications of trkB mRNA were observed in the present study.

The potential for morphological plasticity in the DG is well known with respect to sprouting of mossy fiber axons after lesions or pathologic hyperstimula- tion of the hippocampus. Mossy fibers can grow recurrent collaterals with active neosynaptogenesis into the supragranular zone of the DG after denervation of the granule cells, 21,34'43 after increased cellular activity ~4'5~ and after removal of CA3 target cells. 43'51 More distal sprouting into the supra- and infra-pyramidal layer of CA3 is also observed after partial loss of target cells. 43'48 Although modifications of electri- cal activity were not investigated in this study, the doses of KA employed to generate the hypertrophic response did not induce epileptic behavior. Since KA is known to be an axon-sparing excitotoxin, ~3 it is unlikely that extra-hippocampal fibers afferent to granule cells suffered major damage. Minor damage of callosal fibers due to the injection needle cannot be ruled out. However, experiments performed with different placements of the injection site within the hippocampus gave further indications that the effects


Fig. 6 Temporal distribution of BDNF and trkB mRNA in granule cells of the dentate gyrus following administration of KA into the dorsal hippocampus. Representative autoradiographs of 33P-BDNF and -trkB cRNA hybridization in normal (A, E) and KA-treated (B-D, F) mice. The KA-injected side is on the left. A strong increase in BDNF mRNA is observed after four days (C) and is kept up to 23 weeks (D). (E, F) Despite an apparent increase, levels of trkB mRNA (E: normal, F) two weeks after KA

injection) are not significantly changed. Scale bar = 1 mm.


A

300-

250-

200,

150,

100.

5O 0

o Fj f

, .P! ,

5 10 40 60 80 100

Days post-injection

120

B ~ 4 0 - t ~

~, 36-

~ 32-

~ 28

~ 24

© 20

n T --q:~ Normal ic

Days post-injection

Fig. 7. (A) Quantitation of the time course of~3P-BDNF, -NGFand -neurotrophin-3 cRNA hybridization in the dentate gyrus of KA-treated animals. Values (mean + S.E.M.) are expressed as a percentage of the control side. BDNF mRNA show a bi-phasic increase, with a peak seven days after the administration of KA and a lower but sustained expression after two weeks, n = 6 for each time-point. (B) Quantitation of ~3p-trkB cRNA hybridization in the dentate gyrus of KA-treated mice as a percentage of the control side. Despite a tendency toward an increase, trkB mRNAs are not significantly higher on the KA-injected

side. n = 6 for each time-point.

observed are directly related to KA and not to the loss of target cells. It remains to be demonstrated, however, whether the changes are a direct result of the stimulation of KA receptors on granule cells or a secondary event linked to modifications occurring in the environment and connectivity of dentate neurons. The involvement of L-glutamate in the regulation of developmental processes is controversial. ~ For example, glutamate can promote neurite outgrowth of cerebellar granule cells in early development, 45 but dose-dependent inhibitory effects on neurite elongation and neuronal survival have also been reported? ~ The implication of glutamate receptors with high-affinity kainate sites in growth-related processes is currently not known.

Hippocampal cells express the highest brain levels of neurotrophins, factors of the NGF family. ~7,47 Levels of mRNA for NGF and BDNF are increased in the hippocampus by several experimental paradigms that lead to neuronal activation (for review see Ref. 22). The induction of neuronal activity

by generalized 16a9'29'3° or focal hippocampal seizures, 5° by electrolytic lesion of the DG hilus, 49 and by brief periods of ischemia and hypoglycemic coma 37'52 leads to a dramatic overexpression of the BDNF gene. Increases in BDNF mRNA levels can be mediated via non-N-methyl-D-aspartate 57 and N-methyl-D-aspartate 25'2s glutamate receptors. However, such increases are rapid and transient, and it is not clear if they are related to the permanent changes in axonal architecture of granule cells. Alternatively, they may reflect immediate attempts to survive major extrinsic or intrinsic alterations. In our paradigm, the overexpression of BDNF was delayed, established progressively and was maintained for several weeks, in close correlation with morphological changes. The results point to a different implication of BDNF in the induction and/or maintenance of a developmental program. It is noteworthy that high levels of expression of BDNF are also observed during the normal post-natal maturation period of DG granule cells. 18,37


Although axonal sprouting of undamaged nerves has been shown in the peripheraP 5 and central 54 nervous system to depend upon target-derived N G F , no modifications of N G F m R N A in the D G were observed in this study, with the exception of the transient, already well-described, increase observed within hours following the administration of KA.

Several reports have recently demonstrated a concomitant expression of neurotrophins and their receptors in the CNS raising the possibility of autocrine actions. 8'19'32'33'39'42'5° A similar hypothesis, formulated

as a basis for the induction of mossy fiber sprouting in the DG, 33'5° has been recently validated by the direct colocalization of B D N F and TrkB m R N A after lesion-induced activity. 2'32 In contrast, we have observed no significant modifications of the expression of trkB. Although an initial autocrine/paracrine effect or B D N F cannot be ruled out, the lack of a maintained overexpression of the trkB gene may reveal a mode of action of B D N F

to promote or sustain cell growth that differs from its action leading to axonal sprouting.

CONCLUSION

The present results further extend the capacities for plasticity of the adult mammalian CNS. They show that intrinsic or environmental changes induced by K A may lead to the reinduction of cell growth in adult neurons. Al though the mechanisms involved remain to be determined, the unusual pattern of morphological and molecular changes suggests that they differ from those previously described in other examples of hippocampal plasticity.

Acknowledgements--We are grateful to Dr J. Diamond, Prof. J. Zimmer and Dr M. Peschanski for their critical review of the manuscript and to Regeneron Pharmaceutical Inc. for providing us with BDNF, NGF, NT-3 and trkB cDNAs. This work was supported by grants from the Fondation pour la Recherche M6dicale.

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(Accepted 2 September 1994)

morphogenetic effect of kainate on adult hippocampal neurons associated with a prolonged expression...

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