European Journal of Neuroscience, Vol. 3, pp. 1008-1015 0 European Neuroscience Association 0953-81 &/91 $3.00

Nerve Growth Factor Receptor-immunoreactive Fibres Innervate the Reticular Thalamic Nucleus: Modulation by Nerve Growth Factor Treatment in Neonate, Adult and Aged Rats

M. Fusco’, M. Bentivoglio2, G. Vantini’, D. Guidolin’, P. Polato’ and A. Leon’ ‘Fidia Research Laboratories, Via Ponte della Fabbrica 3/A, 35031 Abano Terme, Padova, Italy 21nstitute of Anatomy, University of Verona, Italy

Key words: thalamus, Alzheimer’s disease, basal forebrain, trophic factor

Abstract

Terminal arborizations expressing nerve growth factor receptor (NGF-R) have been detected with immunohistochemistry in the reticular thalamic nucleus of neonate, adult and aged rats. lntracerebroventricular administration of nerve growth factor (NGF) resulted in a dramatic increase in NGF-R immunoreactivity throughout the lifespan. This effect was paralleled by a concomitant increase in NGF-R immunopositivity in the neurons of the basal forebrain, which was here demonstrated also in aged animals, thus indicating that the NGF-R immunoreactivity within the reticular thalamic nucleus derives in all likelihood from cholinergic neuronal cell bodies of the basal forebrain. Our results demonstrate a prominent ability of NGF to up-regulate its receptors within fibres innervating the reticular thalamic nucleus, and show that this up-regulation of NGF-R is maintained throughout the lifetime. Altogether this indicates that the reticular thalamic nucleus may represent a new, important site of action of endogenous NGF or NGF-like molecules within the brain. In view of the crucial role played by the reticular thalamic nucleus in gating thalamocortical information, the autoregulation of NGF-R in this structure may have important concomitants in both physiological and pathological conditions.

Introduction

Among polypeptide growth factors capable of influencing the survival, differentiation, proper target innervation and function of neural cells during both prenatal and postnatal life, nerve growth factor (NGF) is the best studied and characterized (Levi-Montalcini and Calissano, 1986; Levi-Montalcini, 1987). A physiological, trophic role for NGF in sympathetic and neural crest-derived sensory neurons has been well established, as indicated by the dramatic effects observed after its pharmacological or immunological removal (Levi-Montalcini and Angeletti, 1968; Thoenen and Barde, 1980). In the rat central nervous system (CNS), NGF has been shown to play a physiological role in cholinergic interneurons of the corpus striatum and basal forebrain cells projecting to the hippocampus and neocortex (Gnahn et al., 1983; Mobley et al. , 1985; Vantini et al., 1989). An important feature of the action of NGF in the basal forebrain cholinergic neurons is its capability of modulating the expression of its own receptors (Cavicchioli et al., 1989; Higgins et al . , 1989). Following intracerebroventricular (i.c.v.) administration of NGF marked increases in both receptor protein

and corresponding mRNA were observed in the septa1 area of neonatal and adult rats (Caviocchioli et al . , 1989; Higgins e f al., 1989).

The widespread distribution of NGF, NGF receptor (NGF-R) and the corresponding mRNAs suggests that NGF may play a relevant role in other CNS cell populations (Korsching et al., 1985; Large et a / . . 1986; Shelton and Reichardt, 1986; Whittemore et al . , 1986; Richardson et al . , 1986; Buck et al . , 1988; Yan and Johnson, 1988; Pioro and Cuello, 1990a, b). The thalamus has not yet raised great interest as a potential target of NGF action, although NGF-R immunostaining has been detected in discrete thalamic nuclei during both development (Yan and Johnson, 1988; Eckenstein, 1988) and adulthood (Woolf et al., 1989; Pioro and Cuello. 1990a). We report here that NGF-R-positive fibres, detected by means of the monoclonal antibody 192-IgG (Chandler et al. , 1984; Taniuchi et al., 1986), are present in the reticular thalamic nucleus (Rt) of newborn, adult and aged rats, and that NGF-R-like immunostaining increases throughout the lifespan following i.c.v. administration of NGF. These results are

Correspondence to: Dr M. Fusco, as above

Received I5 February 1991, revised 14 June 1991, accepted 17 June 1991

NGF modulation in reticular thalamic nucleus 1009

discussed in view of the possible physiological and pathological relevance of the Rt as a new potential target for NGF action.

Materials and methods

Materials NGF was purified as the 2.5s subunit from the submaxillary glands of adult male Swiss albino mice (Bocchini and Angeletti, 1969). The biological activity of the isolated NGF, evaluated using embryonic chicken dissociated dorsal root ganglion cells in vitro, was in the range of 0.1 -0.2 ng protein/trophic unit (Varon et al., 1972). For experimental purposes, NGF was dissolved in phosphate-buffered artificial cerebrospinal fluid containing 0.01 % bovine albumin as carrier protein (Williams et a/. , 1986). The antibody used was the 192-IgG, a mouse anti-rat NGF receptor monoclonal antibody (Chandler et al., 1984). The 192-IgG was provided by E. M. Johnson and has been extensively characterized (Taniuchi and Johnson, 1985; Taniuchi et al., 1986). Affinity-purified biotinylated horse anti-mouse IgG (preadsorbed to remove cross-reactivity for rat IgG), normal horse serum and avidin - biotin - peroxidase complex (ABC) peroxidase kits (Vectastain) were purchased from Vector Laboratories (Burlingame, CA). The ME 20.4 IgG was purchased from Amersham (Amersham, UK). Other chemicals were obtained from Sigma (St Louis, MO).

Animals and treatment protocols A total of six aged males (24 months old), eight adult males and six newborn Sprague - Dawley rats were used for immunocytochemical study. Aged rats were selected from a severely impaired population of animals tested by means of the water-maze task (Morris, 1984; Fischer et a/ . , 1987, 1989). All surgical procedures were performed under anaesthesia: hypothermia was used in newborns, whereas the adult and aged rats were anaesthetized with sodium pentobarbital (50 mg/kg, i.m.). In newborns, NGF (5 pg/5 pl) was intracerebro- ventricularly injected on postnatal days 2 , 4 and 6. These animals were sacrificed at postnatal day 8. Newborn control cases received an equal volume of vehicle containing 5 pg of cytochrome c, a protein which has similar physicochemical properties to NGF. In adult and aged rats a cannula was stereotaxically inserted in the lateral right ventricle, permanently fixed, and connected to a mini-osmotic pump (Alzet, model 2001; flow rate 1 pl/h, mean fill volume 200 pl) filled with 50 pg NGF. Control cases received equal amounts of cytochrome c dissolved in the same infusion vehicle. The adult and aged rats were sacrificed on the eighth day following cannula implantation. At the end of the treatment, the NGF remaining in the minipumps (mean residual volume - 30 pl) was still biologically active when assessed in vitro (Skaper and Varon, 1982).

192-lgG immunohistochemistry At the end of treatment all of the rats were again deeply anaesthetized and perfused transcardially with phosphate-buffered saline (pH 7.2) followed by 4 % paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.2). The brains were removed, dissected and postfixed in the same fixative for 2 h at room temperature. Tissue blocks were equilibrated with 30% sucrose in phosphate buffer at 4°C until they sank, frozen in dry-ice and subsequently cut into 40-pm thick serial coronal sections with a freezing sliding microtome. Regularly spaced sections (at 100- 150-pm intervals) through the forebrain and diencephalon of newborn, adult and aged rats were processed for immunohistochemistry .

Serial sections were incubated with 0.7% hydrogen peroxide at room temperature to inhibit endogenous peroxidase. Indirect immuno- histochemistry was carried out using the ABC method (Yan and Johnson, 1988) on floating sections, and affinity-purified rat IgG- absorbed, biotinylated horse anti-mouse IgG were used as secondary antibodies. Sections from cytochrome C- and NGF-treated animals were processed simultaneously in the same incubation solutions. Cytoarchi- tectonic controls were made on a standard Nissl-stained section series of the newborn, adult and aged brain. Controls for the specificity of the immunohistochemical method were run on sample sections through the forebrain, either with no primary antibody or with antibody ME 20.4 IgG, which recognizes the human but not the rat NGF-R (Ross et al., 1984). No immunostaining was detected in these sections.

Computer-assisted image analysis Morphometric and microdensitometric analyses were carried out using an AT-IBAS image analyser (Kontron, Eching, Germany). A high- resolution black-and-white TV camera (Bosch TYK9B) was coupled to an UMSPSO light microscope (Zeiss, Oberkochen, Germany), and a ~ 2 . 5 primary magnification was used. In adults, fifteen sections throughout the extent of the Rt were selected and both immunostaining intensity and stained area estimated as previously described (Zoli et al., 1990) in order to obtain a semiquantitative comparison of antigen contents.

Sections from an NGF-treated adult rat were utilized to achieve a three-dimensional reconstruction of the NGF-R-positive volume by using a stereomicroscope (Olympus SZ40) and the VIDS-3D reconstruction software (AMS, Cambridge, UK). The outlines of the Rt were digitized into the computer along with the outlines of the whole section. The midline and the tangent to the ventral border of the brain were used as reference axes for section-to-section alignment.

Results Immunoreactivity to the NGF-R was detected throughout the extent of the Rt in the control cases treated with cytochrome c. The immunostaining was associated in the Rt with preterminal and terminal arborizations, whereas Rt cell bodies were not immunopositive. In the adult or aged rats, the overall immunostaining of the Rt was relatively light, barely detectable at low-power observation (Figs 1C and 2B). On the other hand, NGF-R immunoreactivity of neuronal processes innervating the Rt appeared relatively intense in the newborn animals (Fig. 1A). In all of these control cases, sparse immunopositive varicose arborizations and puncta were evident when observing the Rt at higher magnification.

The intensity of the immunostaining was markedly enhanced in the Rt in all cases treated with NGF, as compared to the control ones. A dense plexus of neuronal arborization expressing NGF-R immunoreactivity became evident bilaterally in the Rt in all of the NGF- treated cases, independently of the age of the animal, outlining, even at low-power observation, the characteristic crescent-like shape of this structure which surrounds the dorsal thalamus. Clusters of immunoreactive puncta, indicative of terminal ramifications and synaptic enlargements, were frequently observed in the NGF-treated cases around the cell bodies of the Rt. This increase in the immunoreactivity of the Rt neuropil following NGF treatment was dramatic in both the adult and aged rats (compare Fig. 1C and D, and Fig. 2B and E). Such an effect of NGF administration was slightly

1010 NGF modulation in reticular thalamic nucleus

NEWBORN

Frc.1 , Low-power microphotographs of the immunopositivity to the nerve growth factor receptor detected in the reticular thalamic nucleus of rats at birth (A, B) and in adulthood (C, D). A and C illustrate the immunopositivity observed in control cases; B and D show the staining detected in the animals following intracerebroventricular administration of nerve growth factor. Note in all cases the fibre preterminal and terminal-like immunostaining in the reticular nucleus; note that the immunopositivity is higher in control newborn (A) than adult (C) animals. A striking enhancement of the immunostaining is evident after treatment with nerve growth factor (B, D). Scale bars = 100 pm.

less prominent in neonatal animals where, as mentioned above, the immunopositivity of the Rt neuropil was already relatively intense in the control cases (compare Fig. 1A and B).

The morphometric and densitometric analysis performed within the

Rt in the adults (Fig. 3) further confirmed that NGF-R immunoreactivity was well evident throughout the anteroposterior extent of the nucleus after NGF administration (Fig. 3A -D). NGF-R immunostaining appeared diffusely distributed along the dorsoventral axis of the Rt,

NGF modulation in reticular thalamic nucleus 101 1

FIG. 2. Immunostaining of the nerve growth factor receptor observed in the cell bodies and neuropil of the basal forebrain (A, D) and in axonal arborization within the reticular thalamic nucleus (B, C, E. F) of impaired aged rats. in control cases (A-C) and after intracerebroventricular administration of nerve growth factor (D-F). Note that in both structures the immunopositivity is markedly increased in animals treated with nerve growth factor. A dense plexus of terminal arborization, which is hardly visible in control cases (B, C). becomes evident after treatment with nerve growth factor in the reticular nucleus (E, F), consistent with findings observed at birth and in adulthood. Scale bars: A, B, D, E, 100 pm; C, F. 20 pm.

with a prevalence in its most dorsal and ventral sectors (Fig. 3A-C). The image analysis of a control case and an NGF-treated one provided a densitometric documentation of the pronounced enhancement in

NGF-R immunoreactivity expressed in the Rt neuropil after NGF treatment (Fig. 3E).

In agreement with previous observations (Cavicchioli et a/. , 1989),

1012 NGF modulation in reticular thalamic nucleus

FIG. 3. Image analysis of NGF-R immunoreactivity in the reticular thalamic nucleus of adult rats. Sections A-D and E (right panel) are from an NGF-treated rat. Section E (left panel) is from a control case. (A-C) Densitometric analysis of immunostaining in coronal sections at different anteroposterior levels (approximate locations relative to bregma are: A, - 1.8 mm; B, -2.8 mm; C, -3.6 mm; coordinates according to Paxinos and Watson (1986). (D) Three-dimensional reconstruction of reticular nucleus sectors displaying NGF-R-positive fibres. View is from the rostra1 side at 45" lateral tilt. The landmark of the hippocampus has been included as an anatomical reference. (E) Densitometric comparison between immunoreactivity in one sample section through the middle third of the reticular nucleus of a control (left) and an NGF-treated (right) rat. In the densitometric analysis pseudocolours are used to illustrate differences in the grey tones of the immunostaining intensity. Note in A -D that immunoreactivity is distributed throughout the nucleus. Note in E the prominent increase measured after NGF administration.

NGF modulation in reticular thalamic nucleus 1013

NGF administration also induced a prominent increase in NGF-R immunostaining bilaterally in the basal forebrain of newborn and adult animals. A similar effect was also observed in the NGF-treated aged cases, in which NGF-R immunoreactivity was clearly enhanced in the neuronal cell bodies and neuropil of the basal forebrain (compare Fig. 2A and D).

Discussion The expression of NGF-R in neuronal cell bodies and processes has been repeatedly demonstrated in the rat CNS during development (Yan and Johnson, 1988; Eckenstein, 1988), adulthood (Richardson et al., 1986; Pioro and Cuello, 1990a, b) and ageing (Bucker al., 1988; Koh and Loy, 1988). In these studies, NGF-R was consistently detected within the cholinergic neurons of the basal forebrain, where its expression appears to be regulated at both transcriptional and translational levels by NGF itself during development and adulthood (Cavicchioli et al. 1989; Higgins et al., 1989). The ability of NGF to regulate its receptor has also been observed in sympathetic and dorsal root ganglion neurons both in vitro and in vivo (Lindsay et al., 1990; Miller et al., 1991). Nuclear run-off transcription assays suggest that the increase in steady-state NGF receptor mRNA is mediated at the transcriptional level (Miller et al., 1991). The present immunohisto- chemical findings emphasize that the effect of NGF on its receptor is maintained in the basal forebrain of aged animals. In support of the present data, NGF administration was shown to reverse degenerative changes in forebrain cholinergic cell bodies and produce behavioural improvement in behaviourally impaired old rats (Fischer et al., 1987), and to reverse the lesion-induced changes in septa1 cholinergic neurons after fimbrial transection (Montero and Hefti, 1989).

Very few data are available concerning the expression of the NGF-R in the thalamus: transient immunoreactivity to the NGF-R was detected during development in the neuropil of thalamic sensory relay nuclei, such as the ventroposteromedial and lateral geniculate nuclei (Yan and Johnson, 1988; Eckenstein, 1988). Some fibres immunoreactive to the NGF-R were also observed in the Rt of adult rats (Pioro and Cuello, 1990a; Woolf et al., 1989). The present observations extend these data, providing evidence of relatively intense NGF-R immunostaining of fibres innervating the Rt at birth, and of sparse NGF-R-immunopositive innervation of the Rt throughout adulthood and ageing. Furthermore, the present results provide evidence that NGF exerts a prominent up- regulation of the expression of its receptor in the Rt throughout the animal’s lifetime. The NGF molecule is known to initiate its effects upon interaction with its receptor and subsequent retrograde transport of the NGF - NGF-R complex through axons (Johnson and Taniuchi, 1987). Recent immunohistochemical findings indicate that the Rt cells express a pro-NGF precursor molecule (Senut et al., 1990). Taken together with the present study, these data point to the Rt as a previously undetected site of regulation of NGF activity. In relation to this, it is worth recalling that only Rt neurons displayed a regenerative response to peripheral nerve grafts inserted into the rat thalamus (Benfey et al., 1985).

The present observations also strongly argue for a relevant role of NGF in the regulation of the complex function of the Rt. This role could be shared by NGF-like molecules, such as the brain-derived neurotrophic factor and the hippocampus-derived neurotrophic factor (also called neuronotrophin-3 or neurotrophin-3). whose occurrence

in the brain has been recently demonstrated (Maisonpieme et al. , 1990; Hofer er al. , 1990; Rosenthal et al., 1990; Ernfors et al., 1990). However, no detailed localization of their expression in the thalamus is yet available. NGF-like molecules seem to recognize a common low- affinity receptor, while high-affinity receptors would discriminate among NGF, brain-derived neurotrophic factor and hippocampus- derived neurotrophic factor (Rodriguez-TCbar et al . , 1990). In view of this and of the relatively high amounts of NGF used in this study, the possibility that exogenously supplied NGF may exert its effect by interacting with receptors selective for other NGF-like molecules cannot be ruled out.

The fibres which express the NGF-R and innervate the Rt are likely to derive from the neurons of the basal forebrain. The Rt receives afferents from the basal forebrain in several mammalian species (Steriade et al., 1987; Parent et al., 1988; Asanuma, 1989). The basal forebrain neurons projecting to the Rt are represented by both cholinergic and GABAergic cells (Parent et al., 1988; Asanuma and Porter, 1990). Many of the former cells express the NGF-R (Batchelor et al., 1989) and the Rt receives a robust cholinergic innervation (Hallanger and Wainer, 1988). Neuronal cell bodies expressing NGF-R have not been detected in the other structures which provide main input to the Rt, such as the cerebral cortex, thalamus and cholinegic mesopontine cell groups (Pioro and Cuello, 1990a, b; Woolf et al., 1989).

Rt cells are GABAergic (Houser et al. , 1980), and their axons terminate within dorsal thalamic nuclei (Scheibel and Scheibel, 1966). Through their inhibitory action, Rt neurons regulate the firing mode of dorsal thalamic cells, playing the role of a pacemaker of spindle oscillations in the thalamocortical system (Steriade et al. , 1985) and hence in the genesis of the cortical activity underlying the regulation of alertness. Rt cells are located in a strategic position to filter also sensory transmission from the thalamic relay nuclei to the neocortex, and to control the descending influence of the cerebral cortex upon the thalamus through thalamocortical and corticothalamic axon collaterals (Jones, 1985).

NGF-R expression is selectively reduced within the cholinergic neurons of the basal forebrain which exhibit degenerative changes in Alzheimer’s disease (Higgins and Mufson, 1989; Hefti and Mash, 1989). Afferents to the Rt express, in Alzheimer’s disease, the pathological protein Alz-50, with a terminal-like pattern probably localized in the fibres which derive from the basal forebrain (Tourtellotte er al., 1990). This finding suggests that dysregulation of the information processed through the Rt could be involved in Alzheimer’s disease.

The present demonstration that the fibres innervating the Rt are powerfully affected by NGF strongly indicates that this structure is a new potential site of action of endogenous NGF or a NGF-like molecule in the brain. On this basis, NGF could play a role in the physiological and pathological regulation of cortical neurons not only through the direct innervation of the cerebral cortex arising from the basal forebrain, but also through the gate exerted by Rt cells on the activity of thalamocortical neurons.

Acknowledgements We wish to thank Dr E. M. Johnson Jr, for providing the purified 192-IgG. We also thank L. Bigon for NGF purification, and A. Bedeschi and L. Polito for secretarial assistance.

1014 NGF modulation in reticular thalamic nucleus

Abbreviations CNS central nervous system GABA y-aminobutyric acid i.c.v . intracerebroventricular NGF nerve growth factor NGF-R nerve growth factor receptor Rt reticular thalamic nucleus

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