Developmental profile of NGF immunoreactivity in the rat brain: a possible role of NGF in the establishment of cholinergic terminal fields in the hippocampus and cortex

Abstract

In the current investigation, we have examined the developmental profile of nerve growth factor immunoreactivity (NGF-ir) in the postnatal rat. During the first 3 weeks after birth, NGF-ir was observed within the hippocampal mossy fiber region, where it persists throughout adulthood and appeared transiently within three additional zones – the dentate gyrus supragranular zone, the tenia tecta/intermediate lateral septum, and the cingulate/retrosplenial cortex. In all cases, the appearance of NGF-ir progressed in a rostrocaudal pattern over time. A strong correlation was seen between the pattern of NGF-ir and cholinergic innervation in the dentate gyrus supragranular zone, both spatially and temporally, suggesting that NGF may direct the innervation of cholinergic afferents to this region. A spatial correlation was also observed between NGF-ir and cholinergic innervation within the retrosplenial cortex and tenia tecta. With our current techniques, however, we were unable to determine at what point during development the adult-like pattern of cholinergic terminal innervation in these regions occurred and, thus, were not able establish a temporal correlation in these regions. Within the cingulate cortex, there was no evidence suggesting that the developmental appearance of NGF-ir in this region was associated with a specific enhancement of cholinergic innervation. Thus, the results of the current investigation clearly identify the presence of transiently occurring zones of NGF-ir during postnatal CNS development, although defining their exact functional role will require additional investigation.

Introduction

Nerve growth factor (NGF) has been postulated for many years to serve as a target-derived retrograde signal for specific populations of neurons in the peripheral nervous system where it influences neuronal survival and differentiation during critical periods of development. Within the CNS, a similar role of NGF might be inferred for specific populations of neurons in the basal forebrain based upon several lines of evidence. NGF is present in highest concentrations within the innervation targets of basal forebrain cholinergic neurons 28, 36, and cholinergic neurons within the basal forebrain possess both high and low affinity receptors for NGF 4, 20, 25. NGF is specifically bound to these receptors and retrogradely transported from the target territories to the basal forebrain 9, 35. The possibility that NGF might act as a cholinergic differentiation factor has been suggested based on the demonstration that (i) NGF expression in basal forebrain target territories is correlated with choline acetyltransferase (ChAT) activity during postnatal development [29], and (ii) NGF has been shown capable of stimulating ChAT activity in perinatal basal forebrain cholinergic neurons, both in vitro and in vivo 19, 33. Speculation that NGF might also serve as a target-derived chemoattractant for guiding axonal outgrowth from basal forebrain cholinergic neurons to their appropriate targets has also been offered based upon (a) in vitro slice culture studies demonstrating that basal forebrain explants selectively send out AChE-positive fibers toward cortical or hippocampal slices but do not respond similarly to non-target slices 17, 18, and (b) in vivo adult rat regeneration models, whereby NGF both stimulates and directs cholinergic axonal regrowth 21, 22. The ability of NGF to rescue injured basal forebrain cholinergic neurons in selected animal models of neurodegeneration 24, 40has also led to the suggestion that NGF may act as a developmental survival factor for these neurons in much the same way NGF acts upon peripheral sensory and sympathetic neuronal cell populations.

Most of these proposed roles for NGF in the developing basal forebrain cholinergic system, however, have not been supported by recent investigations using null mutations for either the NGF or the high affinity NGF-receptor (TrkA) genes 14, 37. These studies have indicated that (i) NGF is not essential for the survival of central cholinergic neurons of the basal forebrain during development, (ii) basal forebrain neurons develop a cholinergic phenotype in the absence of NGF (although reduced ChAT levels in these animals may suggest cholinergic activity is modulated to some extent by NGF availability), and (iii) basal forebrain cholinergic neurons extend processes toward, and ramify within, their normal cortical and hippocampal targets without NGF being present. Furthermore, studies comparing the developmental onset of NGF gene expression (and protein production) within CNS innervation targets and the time when these targets become innervated by NGF-sensitive fibers have further indicated that target innervation has occurred well before NGF expression has begun 27, 32, 39. This is in marked contrast to what is known for the situation in the developing peripheral nervous system, where NGF expression in a peripheral tissue occurs either before or at the time of its innervation by NGF-sensitive afferents 15, 16. In the hippocampal formation, however, the onset of NGF expression is most tightly correlated with the onset of synaptogenesis 13, 39, raising the possibility that NGF plays a major role in the establishment of synaptic connections.

In previous studies, we have used immunohistochemical techniques to demonstrate the presence of NGF in restricted subregions of the hippocampal formation [8]. In addition, a defined model of sympathetic sprouting into the hippocampal formation has provided evidence for a strict correlation between the tissue regions where NGF is localized and those which the sprouting sympathetic axons will innervate 10, 41. Together, such findings have led to the idea that NGF may topographically define the terminal fields for NGF-sensitive afferents during normal, as well as experimentally induced sprouting. We have extended the use of the sympathetic sprouting model to show that the pattern of experimental sympathetic innervation can be deliberately altered by either additional lesion-induced regions of NGF immunoreactivity [10]or exogenously added NGF [11], thereby confirming that the original distribution of endogenous NGF was critical for defining where the sympathetic fibers would innervate.

Using another experimental model, previous studies have shown that the interruption of entorhinal afferents to the hippocampal formation causes the endogenous cholinergic afferents to undergo synaptic remodeling 12, 30. Using this model, we have demonstrated that concurrent changes occur in the distribution of NGF-ir within the hippocampal formation, again encouraging the view that NGF may be involved in determining where these sprouting fibers will go [7]. Furthermore, blocking the actions of endogenous NGF completely blocks cholinergic sprouting in this model [38]. An interesting facet concerning sprouting of the cholinergic terminals in this model is that the lesion-induced appearance of NGF-ir in the region where sprouting takes place was transient. The appearance of NGF-ir in the deafferented region, which preceded the onset of cholinergic sprouting, was maximal during the time when cholinergic sprouting has been reported to be greatest and declined at later times when cholinergic sprouting has reportedly slowed [7]. Moreover, the lesion-induced NGF-ir would occur even if the cholinergic sprouting was prevented (by removing the cholinergic afferents from the hippocampal formation). This suggested that the NGF is required to establish a new terminal field for the sprouting cholinergic fibers, but may not be required once reorganization is complete.

The apparent ability of topographically restricted regions of NGF (even transiently occurring ones) to establish terminal fields for incoming NGF-sensitive axons has raised the question whether such a mechanism may be directing the distribution of normal endogenous basal forebrain cholinergic afferents within their targets during development. To begin investigating such a question, we have examined the developmental profile of NGF-ir in the postnatal rat.