Fos induction in cortical interneurons during spontaneous wakefulness of rats in a familiar or enriched environment

Abstract

It has been repeatedly reported that Fos is spontaneously induced in several brain structures, including the cerebral cortex, during wakefulness. To ascertain whether cortical interneurons are involved in this state-dependent oscillation of gene regulation, we combined Fos immunocytochemistry with immunostaining of either parvalbumin or calbindin, known markers of cortical interneurons. Immunopositive neurons were examined in the sensorimotor and cingulate cortex. In rats perfused in basal conditions, a minor proportion (around 8%) of Fos-immunoreactive neurons in the parietal cortex were also parvalbumin- or calbindin-immunoreactive; these double immunostained cells accounted for 13% of the parvalbumin- and 34% of the calbindin-labeled neurons. Colocalization of Fos with either calcium-binding protein was instead not observed in the cingulate cortex. In rats stimulated by novel environmental cues during the period of wakefulness preceding perfusion, Fos-positive neurons increased markedly relative to unstimulated animals, and involved the majority of the calbindin- or parvalbumin-labeled cell populations (60–75% and over 95%, respectively). In the neuronal populations in which Fos was induced by exposure to the enriched environment, the proportion of calbindin- and parvalbumin-labeled cells was larger than in the unstimulated cases, and the increment was statistically significant in the cingulate cortex. The results demonstrate that Fos induction occurring in the cortex during undisturbed wakefulness in a familiar environment involves a minor proportion of interneurons. Furthermore, the findings indicate that the addition of novel environmental stimuli results in an increase of Fos-expressing neurons whose recruitment, at least in the cingulate cortex, involves a higher proportion of interneurons than of projection neurons.

Introduction

The expression of the immediate early gene c-fos has been reported to oscillate during the spontaneous sleep/wakefulness cycle in neuronal populations of the brain (see [1] for review). In particular, it has been repeatedly described that c-fos mRNA and its protein product Fos are induced during wakefulness in the rat cerebral cortex 7, 8, 18. These and other studies have provided evidence that gene expression fluctuates considerably in relation to the circadian variation of functional states, as well as to the type of activity that the animal is engaged in 15, 19. However, it remains to be determined whether certain cortical cell populations are preferentially involved in such oscillation.

In the present study, we aimed at a characterization of cortical neurons involved in behavioral state-dependent Fos induction. In particular, we sought to ascertain whether Fos is induced during wakefulness in interneurons of the cingulate and parietal cortex, and we examined this issue with a double immunocytochemical approach.

In the cerebral cortex, short-axon neurons comprise heterogeneous subsets of inhibitory GABAergic cells, which represent approximately 15–30% of the total neuronal cell population (see [6] for review). On the other hand, cortical projection neurons, which are excitatory and utilize amino acids as neurotransmitters, constitute the largest component (approximately 70–85%) of the total population of cortical neurons [6]. A number of different neuroactive molecules, including neuropeptides and calcium-binding proteins of the EF-hand family, have been shown to be co-localized with GABA in cortical interneurons. In particular, cells immunostained by the calcium-binding proteins parvalbumin (Pv) or calbindin D-28k (Cb) represent, taken together, more than 90% of cortical GABAergic interneurons, although with areal and species variations [6]. In the rat cortex, 70% of the GABAergic cells contain Pv [4], while Cb-positive neurons include a much lower percentage of cortical nonpyramidal cells 5, 6. Pv- and Cb-containing cells mostly represent discrete subpopulations of cortical interneurons, and co-localization of the two proteins in the rat somatosensory cortex can be demonstrated in a very limited subset of neurons [20]. In the present study, we selected Pv and Cb immunoreactivity, which is fully compatible with Fos immunostaining (at variance with GABA immunocytochemistry, which requires a different perfusion recipe), to characterize populations of interneurons in the cerebral cortex of rats perfused during wakefulness, both in the absence of specific stimulation and after introducing novel environmental cues that trigger exploratory behavior.