The expression pattern of somatostatin and calretinin by postnatal hippocampal interneurons is regulated by activity-dependent and -independent determinants

Pergamon

PII:

Neuroscience Vol. 80, No. 1, pp. 79–88, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+0.00 S0306-4522(97)00134-6

THE EXPRESSION PATTERN OF SOMATOSTATIN AND CALRETININ BY POSTNATAL HIPPOCAMPAL INTERNEURONS IS REGULATED BY ACTIVITY-DEPENDENT AND -INDEPENDENT DETERMINANTS S. MARTY* and B. ONTE u NIENTE INSERM Unite´ 421, Faculte´ de Me´decine, 8 rue du Ge´ne´ral Sarrail, 94010 Cre´teil, France Abstract––Hippocampal interneurons form distinct populations identified on the basis of their projection pattern and neurochemical characteristics, which includes the expression of specific neuropeptides and/or calcium-binding proteins. The neurochemical maturation of hippocampal interneurons is largely a postnatal event, and factors which govern this maturation are presently unknown. Using slice cultures, we have investigated the role of neuronal activity in regulating the expression of somatostatin and calretinin during the postnatal maturation of hippocampal interneurons. Blocking inhibitory activity with bicuculline, or excitatory activity with 6,7-dinitroquinoxaline-2,3-dione, for 14 days in slice cultures from seven-day-old rat increased and decreased, respectively, the number of somatostatin-immunoreactive neurons. Withdrawal of the blocking agents resulted in a reversal of the effects on somatostatin immunoreactivity. In addition, bicuculline slightly increased the number of calretinin-positive neurons, while 6,7-dinitroquinoxaline-2,3-dione exerted no effect. However, bicuculline and 6,7-dinitroquinoxaline2,3-dione markedly increased and decreased, respectively, the number of calretinin-labelled axons. Despite activity-linked modifications of immunoreactivity levels, no change in the organotypic location of somatostatin-labelled neurons was observed, whatever the treatment. Double labelling studies demonstrated that somatostatin and calretinin were expressed by different neurons, even when the number of labelled cells was highly increased. These results show that the levels of expression of somatostatin and calretinin in maturing hippocampal interneurons are tuned to the endogenous balance of excitatory and inhibitory activity. In contrast, the neurochemical specificity of each subtype of interneurons does not depend upon variations in neuronal activity. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: rat, Ammon’s horn, slice culture, GABAergic neurons, neuropeptides, calcium-binding proteins.

The Ammon’s horn contains pyramidal neurons which use glutamate as a neurotransmitter, and nonpyramidal neurons which contain the inhibitory neurotransmitter GABA. GABAergic interneurons can be subdivided on the basis of their neurochemical characteristics, such as the expression of various neuropeptides and/or calcium-binding proteins.2,15,19 Interneurons with specific neurochemical phenotypes are also characterized by distinct physiological and anatomical properties. For instance, the calciumbinding proteins parvalbumin, calbindin and calretinin are contained with little to no overlap in interneurons with distinct axonal projections.12,14,19,28 While hippocampal interneurons are generated prenatally,5,29 their neurochemical maturation largely extends over the postnatal period. This postnatal maturation involves not only the expression of the main neurotransmitter GABA,25,27 but also of

specific neuropeptides or calcium-binding proteins. The number of interneurons containing somatostatin or neuropeptide Y increases from birth to reach adult levels at about the end of the third postnatal week,23,33 while parvalbumin expression starts only at the end of the first postnatal week.7 Factors which govern the expression of specific neuropeptides or calcium-binding proteins in maturing interneurons are presently unknown. Experiments using injections of retroviruses have shown that lineage is not responsible for the expression of a specific calcium-binding protein, indicating the crucial involvement of environmental cues.20 Among environmental cues, several observations suggest that neuronal activity could be responsible for the expression of neuropeptides or calcium-binding proteins in hippocampal interneurons. As with interneuronal phenotype, neuronal activity in the hippocampus matures during the postnatal period. During the first postnatal week, GABA is responsible for ongoing depolarizing activity, before it switches to its classical hyperpolarizing activity in parallel with the

*To whom correspondence should be addressed. Abbreviations: DNQX, 6,7-dinitroquinoxaline-2,3-dione; NMDA, N-methyl--aspartate. 79

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Modulation of interneuronal phenotype by activity Table 1. Effects of bicuculline or DNQX treatment on the number of calretinin-immunoreactive neurons in the stratum oriens Treatment

Control

Treated

P value

Bicuculline DNQX

100&1 100&7

117&3 107&7

0.006 0.514

Results are presented as percentages of controls. Bicuculline induced a 17% increase in the number of labelled cells, while DNQX exerted no effect.

development of glutamatergic transmission.6,9,13 In addition, a depolarizing stimulus increases the expression of somatostatin or neuropeptide Y by cortical interneurons developing in vitro.16,32 On the other hand, the fact that neurochemical characteristics displayed by subgroups of interneurons are linked to distinct afferent and efferent connections suggests that cell–cell interactions more specific than activity may determine the neurochemical fate of interneurons. In order to evaluate the role of neuronal activity in driving the neurochemical phenotype of postnatal hippocampal neurons, the endogenous balance of excitatory and inhibitory activity was manipulated within hippocampal slice cultures, and the effects of alterating neuronal activity on somatostatin and calretinin expressions were observed. Slices were taken from seven-day-old rat hippocampus, when non-N-methyl--aspartate (NMDA) glutamatergic transmission has just been established, while GABAergic connections become inhibitory.6,9,13 The slices were cultured for two weeks, since during this period of time maturation of both inhibitory and excitatory transmission occurs similarly to the in vivo situation.8,22,31 The relevance of somatostatin and calretinin for the purpose of this study lies in the fact that these two markers are expressed in the adult hippocampus by interneurons differing in their projection sites, but which are spatially intermingled.1,2,12 Hence, the cell specificity of expression of these markers in the slices can be studied by examining whether or not they co-localize in the same neurons. EXPERIMENTAL PROCEDURES

Slice culture Cultures of hippocampal slices were prepared using a modified version of the method of Stoppini et al.30 Sevenday-old Wistar rats (Janvier) were decapitated and their brains rapidly removed. Under sterile conditions, hippocampi were dissected in sodium phosphate buffer with 0.9%

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NaCl (0.1 M; pH 7.4). Slices were cut at 400 µm-thickness, perpendicular to the septotemporal axis of the hippocampus, using a McIllwain tissue chopper (Mickle Laboratory, England). Hippocampal slices were transferred into the culture medium, separated and transferred on to Millicell-CM membranes (Millipore, France). A total of 12 adjacent slices were obtained per brain. Adjacent slices were transferred onto different Millicell in order to compare the effects of the treatments with control adjacent sections. The Millicell membranes were kept in 6-well plates, above 750 µl of defined medium. The medium consisted of minimum essential medium (Gibco 11012-010, France), 1% -glucose, 5 mM Tris–HCl, 100 µg/ml bovine serum albumin, 100 µg/ml transferrin, 16 ng/ml putrescin, 40 ng/ml N-selenium, 30 ng/ml tri-iodothyronin, 5 µg/ml insulin and 60 ng/ml progesterone. All chemicals were purchased from Sigma (France). Slices were incubated at 37)C in 5% CO2. The medium was exchanged every second day during the first week in culture, and every third day thereafter. Pharmacological treatments In order to manipulate neuronal activity in the slices, hippocampal slices were transferred into medium containing either 10 µM of the GABAA receptors blocker bicuculline (Sigma) or 20 µM of the non-NMDA glutamate receptors blocker 6,7-dinitroquinoxaline-2,3-dione (DNQX; RBI, Bioblock Scientific, France) immediately after the slices were cut. Immunohistochemistry After fixation for 4 h in 4% paraformaldehyde in sodium phosphate buffer, slices were rinsed several times in phosphate-buffered saline and incubated for 30 min in phosphate buffer containing 1% Triton X-100 and 3% normal goat serum (Gibco). Slices were then incubated overnight at 4)C with antibodies raised against somatostatin (1:1000, gift from Dr J. Epelbaum21) or calretinin (1:5000, Swant, Switzerland) diluted in sodium phosphate buffer containing 1% Triton X-100 and 1% normal goat serum. After washes in sodium phosphate buffer, slices were incubated for 1 h with a solution containing biotinylated anti-rabbit IgG antisera (1:100; Vector Labs, Biosys, France), 1% Triton X-100 and 1% normal goat serum in sodium phosphate buffer. After several washes in sodium phosphate buffer, slices were incubated for 1 h with an avidin–biotin– horseradish peroxidase complex (1:100; Vector Labs) in sodium phosphate buffer containing 1% Triton X-100. Staining was developed with a sodium phosphate buffer solution containing 0.05% diaminobenzidine (Sigma) and 0.01% hydrogen peroxide. Slices were rinsed in phosphate buffer, mounted onto gelatin-coated slides, air dried before clearing in toluene and coverslipped in Permount (Fisher Scientific, Euromedex, France). For double-immunolabelling, immunostaining for somatostatin was performed as described above, except that the staining was developed in Tris buffer (0.05 M; pH 7.4) containing 0.05% diaminobenzidine (Sigma), 0.01% hydrogen peroxide and 0.45% nickel–ammonium sulphate (Nakarai Chemicals, Japan). After washes in sodium phosphate buffer, slices were incubated in a sodium phosphate buffer solution containing 0.03% hydrogen peroxide for 30 min, washed in sodium phosphate buffer, and immunostained for calretinin as described above. Control experiments were performed by omitting primary antibodies.

Fig. 1. Calretinin immunostaining in hippocampal slice culture 14 days after explantation. A) Calretininimmunoreactive neurons and processes are randomly distributed throughout the slice. B) Calretininimmunoreactive cell bodies bear smooth and beaded dendrites. C) Calretinin-immunoreactive axons run parallel to the stratum pyramidale at the stratum oriens/alveus border (the brackets delineate this axonal bundle). D) Calretinin-immunoreactive varicosities (arrowheads) in close apposition with a neuron less intensely stained (star). alv, alveus; so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum. Scale bar=200 µm in A, 60 µm in B and C, 20 µm in D.

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Fig. 2. Effects of bicuculline or DNQX treatment on the number of calretinin-immunoreactive axons at the stratum oriens/alveus border. A) Control slice. B) Bicuculline-treated slice. C) DNQX-treated slice. The brackets delineate the axonal bundle. Note the increase in number of labelled axons after bicuculline treatment, and the decrease in number after DNQX treatment. alv, alveus; so, stratum oriens. Scale bar=50 µm.

Cell counts After immunohistochemistry, the total number of immunopositive cells was counted in the stratum oriens of the CA3 and CA1 fields as described previously.17 In brief, a total of 12 adjacent sections were obtained per brain. All the slices survived and were successfully stained. For each treatment, counts were performed blindly in slices taken from three animals, with three control and three adjacent treated slices analysed per animal. The same was done to analyse withdrawal of the blocking agents. The mean of the values per animal was calculated and statistical analysis was performed with unpaired Student’s t-test. Results were verified in at least two independent experiments. About 80 somatostatin-immunoreactive cells and 70 calretininimmunoreactive cells per slice were observed in control slices after 14 days in culture. RESULTS

Hippocampal slices retained their organotypic organization over the 14 days of explantation. Layers of the dentate gyrus granule cells and pyramidal cells were recognizable and retained their spatial relationships. However, the lately-generated infrapyramidal blade of the dentate gyrus spread, and it was not possible to identify it as a distinct cell layer. After two days in culture, flattening of the slices became apparent and this process continued during the following days resulting in a broadening of the abovementioned cell layers. Thinning of the stratum oriens

permitted good staining of the neurons. No major difference was observed in organotypic features between control and treated slices, with respect to either the size of the slices or their configuration. Effects of alteration of neuronal activity on calretinin immunostaining Calretinin-positive cells were observed throughout the hippocampus, with dendrites extending most often radially, with the exception of the stratum oriens where dendrites of calretinin-labelled neurons extended more parallel to the pyramidal cell layer (Fig. 1A). The immunostaining displayed a broad range of intensities, from faint, just above background, and restricted to the cell bodies, to strong, including long, smooth and sometimes beaded dendrites (Fig. 1B). Axonal processes were also present throughout the slices, distinguishable from dendrites by their thin caliber, the absence of tapering and the presence of varicosities along their course. Axonal processes were found at higher density in the stratum oriens/alveus border where they ran parallel to the pyramidal cell layer (Fig. 1C). Calretinin-positive axonal processes were sometimes observed closely apposed to the soma or dendrites of calretininpositive neurons (Fig. 1D).

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Fig. 3. Somatostatin immunostaining in hippocampal slice culture 14 days after explantation. A) Control slice. B) Bicuculline-treated slice. Note the increase in number of labelled cells after bicuculline treatment. The labelled cells were found mainly in the stratum oriens. so, stratum oriens; sp, stratum pyramidale. Scale bar=250 µm.

After 14 days of bicuculline treatment, the number of calretinin-labelled cells had slightly increased (17&3%) when compared with adjacent control slices (Table 1). The same range of intensity, from faintly to strongly stained cells, was found in experimental conditions and in control slices. However, bicuculline treatment resulted in a marked increase of the number of calretinin-positive axons, which was particularly obvious at the stratum oriens/alveus border, where axonal processes are found at a higher density (Fig. 2A,B). DNQX treatment did not decrease the number of calretinin-immunoreactive neurons (Table 1), and neuronal cell bodies remained strongly labelled (Fig. 2C). However, DNQX treatment markedly decreased the number of calretinin-positive axons (Fig. 2C). Effects of alteration of neuronal activity on somatostatin immunostaining In contrast to calretinin-positive cells that were randomly distributed throughout the layers,

somatostatin-immunoreactive cells presented a specific distribution in the slices. Somatostatinlabelled cells were present mostly in the stratum oriens, with very few cells in the pyramidal cell layer or in the stratum radiatum (Fig. 3A). Somatostatin immunostaining appeared punctiform, and thus different from the homogeneous staining of the cytoplasm in cells immunostained for calretinin (Fig. 4A). Immunostaining of the proximal parts of neurites was sometimes observed. After 14 days of bicuculline treatment, the number and staining intensity of somatostatin-labelled cells strongly increased when compared with adjacent control slices. A 62&8% increase in the number of labelled cells was observed (Fig. 5A). The staining of the cell bodies was more pronounced than in control slices (Fig. 4B). Neuritic labelling was more often observed, and neurites were labelled over a longer distance. However, the localization of the labelled cells remained identical to control, with a high density of immunoreactive cells in the stratum oriens, and few cells in stratum pyramidale and stratum

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Fig. 4. Effects of bicuculline or DNQX treatment on somatostatin-immunoreactive neurons in the stratum oriens. A) Control slice. B) Bicuculline-treated slice. C) DNQX-treated slice. Note the increase in staining intensity of cells after bicuculline treatment, and the opposite effect after DNQX treatment. Scale bar=50 µm.

radiatum (Fig. 3B). DNQX treatment resulted in a dramatic decrease in somatostatin immunostaining. The number of labelled cells was reduced by 38&1% when compared with control slices (Fig. 5A). The labelling was less intense, and neuritic labelling was scarce (Fig. 4C). Double immunostaining for somatostatin and calretinin After double immunostaining for somatostatin and calretinin, neurons positive for either calretinin or somatostatin were found intermingled in the stratum oriens (Fig. 6). About 200 labelled neurons in control slices and 400 labelled neurons in bicuculline-treated slices were examined for a possible co-localization of somatostatin and calretinin. No co-localization was found. Reversal of the effects After two weeks of treatment with either bicuculline or DNQX, the slices were returned to control medium for an additional week. This withdrawal of the blocking agents resulted in a reversal of the effects of either bicuculline or DNQX on the number of somatostatin-immunoreactive neurons (Fig. 5B). DISCUSSION

The aim of this study was to evaluate the influence of neuronal activity in regulating the expression of

the neuropeptide somatostatin and the calciumbinding protein calretinin in hippocampal GABAergic interneurons during their postnatal maturation. For this purpose, neuronal activity was manipulated for 14 days in hippocampal slice cultures from seven-day-old rats, and the effects on somatostatin and calretinin immunoreactivities were investigated. Activity-related modifications of immunostaining revealed that the interplay between excitatory and inhibitory neuronal activity regulates the levels of expression of somatostatin and calretinin. However, interneurons retain their specific neurochemical phenotype whatever the state of activity, showing that the ability of interneurons to express a particular phenotype in the postnatal hippocampus is specified by determinants independent of neuronal activity. The level of somatostatin and calretinin expression is tuned to the endogenous excitatory/inhibitory balance After the first postnatal week, glutamate mediates depolarizing activity in the hippocampus, while GABAA receptor activation exerts hyperpolarizing effects.6,13 Thus, both GABAergic and glutamatergic transmissions are likely candidates to affect the phenotype of GABAergic neurons. In agreement with this hypothesis, blockade of inhibitory, GABAA receptor-mediated synaptic transmission with bicuculline strongly up-regulated the number of

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detect than changes in the level of calretinin staining, which is diffusely distributed throughout the neuronal cytoplasm. The increase in number of calretinincontaining cell bodies and axons after bicuculline treatment suggests that, as is the case for somatostatin content, a modulation of calretinin levels actually occurs in response to changes in neuronal activity. Determinants independent of neuronal activity define the potential neurochemical characteristics of interneurons

Fig. 5. Effects of bicuculline or DNQX treatment on the number of somatostatin-immunoreactive neurons in the stratum oriens. Results are presented as percentages of controls (white bars). A) Bicuculline induced a 60% increase in the number of labelled cells, while DNQX induced a 40% decrease in the number of labelled cells. B) After reversal of either bicuculline or DNQX treatment, no significant differences in the number of labelled cells were observed between control and treated slices.

somatostatin-containing neurons, while blockade of non-NMDA glutamate receptor-mediated excitatory synaptic transmission with DNQX exerted opposite effects, with a striking decrease in the immunoreactivity for somatostatin. Withdrawal of bicuculline or DNQX resulted in reversal of the effects, indicating that these effects reflect a modulation of expression of the neurochemical content rather than a regulation of neuronal survival. Calretinin-containing interneurons displayed a different response. While bicuculline slightly increased the number of immunopositive neurons, DNQX exerted no effect. It is noteworthy that, in contrast to limited modifications observed in the somatic compartment, activity dramatically increased the number of calretinin-labelled axons. It is not clear whether this reflects structural changes of axonal arborizations, or a modulation of calretinin levels in preexisting axons. Although variations of the number of inhibitory synapses have been described after manipulation of neuronal activity in several models,18,26 further investigations are needed to determine whether this applies to our paradigm. Furthermore, absence of modifications in the number of calretinincontaining neurons after DNQX treatment may result from the distribution of immunoreaction product. The punctate labelling of somatostatin immunohistochemistry is consistent with a vesicular storage of the neuropeptide. Changes in the level of this discrete and scarce labelling are likely to be easier to

Deafferentation of the hippocampus due to the slicing protocol and blockade of glutamatergic transmission by DNQX did not abolish immunostaining for somatostatin and calretinin, indicating that a basal expression of these markers occurs even in the absence of excitatory activity. Similarly, it has been recently reported that removal of one set of excitatory afferents delays, but does not prevent, the occurrence of a phenotypic shift in calcium-binding protein expression in developing non-pyramidal neurons in the rat barrel field cortex.3,4 In the adult hippocampus, calretinin-containing GABAergic neurons are present in all strata,11 while somatostatin-positive neurons are found essentially in the stratum oriens, with few cells in the stratum pyramidale and stratum radiatum.15 A similarly restricted distribution of somatostatin in long-term hippocampal slice cultures has been previously reported.10 The same differential distribution of calretinin and somatostatin was found in the slices up to two weeks after explantation. Since this distribution persisted whatever the treatment imposed, the basal expression of somatostatin within a given set of neurons is a property independent of neuronal activity. In addition, as shown by doublelabelling experiments, somatostatin and calretinin remained expressed by different neurons. This absence of ectopic expression points to another property of somatostatin- or calretinin-positive interneurons in the postnatal hippocampus, which is their inability to express the other protein. Lack of co-localization of somatostatin and calretinin questions the nature of these interneurons able to respond to increased activity by an increased synthesis of somatostatin. The other types of interneurons contain parvalbumin or calbindin, and about half of the hippocampal GABAergic neurons do not contain detectable levels of calcium-binding proteins.19 Since parvalbumin-positive basket or axoaxonic interneurons are located mainly within the stratum pyramidale, expression of somatostatin is unlikely to occur in these neurons. However, somatostatin co-localizes partially with calbindin,24 and more calbindin-immunoreactive neurons could be induced to express somatostatin. GABAergic neurons which do not express calcium-binding proteins may also be induced to express detectable levels of somatostatin.

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Fig. 6. Double immunostaining for calretinin and somatostatin. Somatostatin immunoreactivity was revealed using nickel-intensified diaminobenzidine as chromogen (black reaction product), while calretinin immunoreactivity was revealed using diaminobenzidine (brown reaction product). A) Control slice. B) Bicuculline-treated slice. Note the absence of co-localization of somatostatin and calretinin. Scale bar=20 µm.

CONCLUSION

Altogether, these results indicate that determinants other than neuronal activity specify the capability of pools of interneurons to express particular neurochemical phenotypes. The nature of these determinants is currently unknown. Given the specificity of expression of somatostatin as early as postnatal day 1,23 these determinants should start to act early in development. Experiments using retrovirus injection during embryonic development have shown that lineage does not control the expression of specific calcium-binding proteins.20 If neuronal activity early in development is involved, our results indicate that it specifies even those neurons which start to express particular neuropeptides later on during development. Cell–cell interactions other than neuronal activity are likely to be good candidates to determine specific subgroups of GABAergic neurons. The difference in expression of neuropeptides or calciumbinding proteins in the adult hippocampus is corre-

lated with a difference in target specificity. Calretininimmunoreactive neurons were recently found to be a new class of inhibitory cells specialized in the control of other inhibitory neurons, including somatostatinpositive neurons identified by their expression of type 1 metabotropic glutamate receptor.1,2,12 Intracellular injections of biocytin revealed that presumptive somatostatinergic neurons project to the stratum lacunosum moleculare where they are suitably positioned to control afferent activity from the entorhinal cortex.28 How this target specificity emerges during development in relation to characteristic neurochemical properties remains to be elucidated. Acknowledgements—We thank Dr Marc Peschanski and Dr Alexander Rabchevsky for critical reading of the manuscript. We are grateful to Dr Jacques Epelbaum for kindly providing antisera against somatostatin. Serge Marty is supported by a grant from Fondation pour la Recherche Me´dicale.

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