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International Journal of Morphology

On-line version ISSN 0717-9502

Int. J. Morphol. vol.21 no.3 Temuco  2003 

Int. J. Morphol., 21(3):181-189, 2003.


*Oscar Inzunza; *Hermes Bravo & **Víctor Fernández 

INZUNZA, O.; BRAVO, H. & FERNÁNDEZ, V. Environmental enrichment during early postnatal development decreases parvalbumin expression in the rat somatosensory cortex. Int. J. Morphol., 21(3):181-189, 2003.

SUMMARY: In the rat brain, parvalbumin (PV) expression starts on postnatal day 8 and comprises a heterogeneous population of nonpyramidal GABAergic neurons. In the present work, an immunohistochemical study was done on control and experimental rats submitted to enriched environmental conditions between postnatal days 3 to 18 or 3 to 24. Counts of PV+ neurons were made in the dorsomedial and in the ventrolateral regions of the somatosensory cortex.

In control animals, PV+ neurons reached a peak on day 24 declining towards day 120. In these rats a peculiar distribution pattern was detected in which immunoreactive neurons are more numerous in the dorsomedial than ventrolateral regions as well in infragranular than supragranular layers and in posterior regions than anterior ones. Differences observed in these three dimensions were well established on day 24. Rats exposed to the enriched environment from day 3 to day 24 show a reduction (26%) in the number of PV+ neurons. The effects of the enrichment persisted for at least 12 days since animals submitted to the enriched condition from day 3 to day 18 and sacrificed on day 30 present a similar reduction (29%) in the number of PV immunoreactive neurons.

Environmental enrichment induces a significant reduction of PV+ neurons but the overall distribution is retained. This finding suggests some degree of stability in the expression of PV in the rat somatosensory cortex.

KEY WORDS: 1. Neuropeptide PV; 2. Cerebral cortex; 3. Polysensorial stimulation; 4.Rodents; 5. Immunohistochemistry. 


It is well known that environmental enrichment induces changes in the rat brain which include an increase in the thickness of the cerebral cortex (Walsh et al., 1972), a dendritic expansion (Greenough & Volkman 1973), a decrease in the number of neurons and an increase in somata size (Diamond et al., 1964), and an increase in the density of oligodendrocytes (Diamond et al. 1966; Szeligo and Leblond, 1977). The morphological changes induced by exposing animals to environments rich in sensory stimuli do not occur uniformly throughout the brain (Walsh & Cummings 1975) and are more pronounced when the environmental manipulation is induced during the critical period of cortical development (term when the bulk of the dendritic network develops), which corresponds to the first three weeks of life (Venable et al., 1989 and Fernández et al., 1997). The cortical changes seem to be the basis for the improved behavior in terms of exploring attitude, task resolution and learning detected on young rats subjected to environmental enrichment (Gard et al., 1967; Renner & Rosenzweig, 1986 and Venable et al., 1988).

Parvalbumin (PV), a calcium-binding protein, seems to play an important role in neuronal homeostasis (Persechini et al., 1989; van Brederode et al., 1991), differentiation (Heizmann & Hunzinker 1990; Baimbridge et al., 1992), excitability (Braun et al., 1985; Celio, 1986; 1990), and protective processes (Morrison et al., 1988; Nitsch et al., 1989 and Sloviter, 1989).

In the rat brain, PV positive (PV+) cells comprise a heterogeneous population of nonpyramidal GABAergic neurons (Celio 1986; 1990). PV+ somata are found in all layers of the neocortex except layer I, and are more numerous in the somatosensory than in the motor cortex (van Brederode et al. 1991). PV inmunoreactivity in cortical neurons first appears on postnatal day 8 (Soriano et al. 1992). During the second postnatal week, a high percentage of these neurons also express calbindin. In this respect, some evidence indicates that calbindin expression in non pyramidal neurons decreases during the second and third postnatal weeks in parallel with the onset of PV immunoreactivity (Alcantara et al. 1993). Moreover, most PV+ cells seem to be previously calbindin+ neurons (Alcantara et al. 1996). The adult distribution pattern of PV+ neurons appears at the end of the first postnatal month.

PV inmunoreactive expression in the neocortex of the rat is coincidental with the appearance of inhibitory synapses (Miller 1986), inhibitory postsynaptic potentials (Luhmann and Prince 1991) and the refinement of cortical intrinsic circuits (Fox 1992). Moreover, a positive correlation between cytochrome oxidase staining and PV inmunoreactivity in the sensorimotor cortex of the rat has been described (van Brederode et al. 1991), suggesting that PV is a marker for metabolically active groups of neurons. These properties raise the posibility that PV expression may be affected by environmental enrichment during development.

Despite the extensive information available on cerebral cortex neurochemistry (for review see: Emson and Lindvall 1979; Parnavelas and McDonald 1983; Parnavelas et al. 1988), few papers address the interaction between the environment and chemically identified cortical neurons (Zolman and Morimoto 1962; Brown 1971; Bennett et al. 1974; Walsh and Cummings 1976). In this report, we analyze the cortical expression of PV in rats subjected to environmental enrichment during an early period of postnatal development. Our results expose the quantitative distribution of PV+ neurons observed in the supragranular and infragranular layers of the somatosensory cortex. Additionally, we correlate the changes in the overall distribution of PV+ neurons induced by environmental enrichment with the tridimensional gradient of cortical maturation (Smart 1983; Miller 1988).


Animals. Male Sprague-Dawley albino rats, born on the same day, were randomly assigned to four control groups (P18, P24 P30 and P120) or two enriched environmental groups: enriched condition (EC24) and enriched condition-persistence (EP18-30). Each group had 8 pups per nurse.

Environmental enrichment. From postnatal day 3, pups in the enriched environment group were exposed to three, 30 minute long, daily sessions of free exploration in a 70x70x40 cm wire mesh play box furnished with a large variety of objects which were changed or rearranged daily. These included running wheels, tunnels, platforms, ladders, sand boxes, balls, rattle, leafy plants, long grass, flowers, tree branches, sawdust, furry and rough surfaces, and toys of different sizes and textures (wood, plastic and metal). Recorded music was also provided. In each session, the pups underwent gentle handling to induce tactile stimulation for 2 min. before each testing/training session, while 24 small lights flashed intermittently. Afterwards, the pups were gently placed in a water bath at 37C for at least 25 seconds during which time they would swim. Rats in the control groups were handled only for routine maintenance. All animals were housed socially in standard laboratory cages. The animals were raised on a 12/12 hours dark-light cycle, with free access to food and water, at controlled temperature (21 + 1,2 C) and relative humidity (50 + 5%).

The enriched condition group EC24 was sacrificed on postnatal day 24. The enriched condition-persistence group EP18-30 was subjected to the stimulated condition described above until postnatal day 18 and then left without enrichment to be sacrificed on postnatal day 30. Control groups were sacrificed on postnatal days 18 (P18), 24 (P24), 30 (P30) and 120 (P120).

Technical procedure. At each age, animals were deeply anesthetized with sodium pentobarbital (6 mg/100 gr body weight) and perfused through the left ventricle with saline followed by 4% paraformaldehide in phosphate buffer. After perfusion the brains were removed from the skull, postfixed in the same solution overnight, at 4 C and then cryoprotected with 30 % sucrose in phosphate buffer. Brains were frozen and cut in the coronal plane every 50 µm intervals on a sliding microtome. Free floating sections were incubated (4 C, 12-24 h.) with the anti PV monoclonal antibody (Sigma clone PA-235) diluted (1:10000) in phosphate buffer containing 5% normal rabbit serum. Sections were subsequently incubated for 1 h. in biotinylated rabbit anti-mouse IgG antibody (1:200), and avidin-biotin peroxidase complex (Vectastain ABC kit, Vector; dilution 1:100) for 1 h. Peroxidase was developed with diaminobenzidine. The sections were mounted, counterstained with cresyl violet and coverslipped. False positive immunoreactivity was controlled, as usual, by incubating some sections without the primary antibody. Sections were processed under identical conditions, in order to get data as comparable as possible.

Determination of number and distribution of PV+ neurons. Twenty four sections in each experiment were selected for counting PV+ neurons in the somatosensory cortex (Sm). These sections were localized between the anterior commisure and the anterior aspect of the hippocampus (according to the Stereotaxis Atlas of Sherwood and Timiras 1970, this region is located from A = 6.2 mm to A = 4.1 mm for 21-day-old rats and from A = 7.0 to A = 5.0 for 39-day-old rats). Counts were made bilaterally in two vertical strips of cortex, 240 µm wide, from layer I to VI. The strips of cortex were situated in the dorsomedial region of the somatosensory area which includes the disgranular portion of SmI (van Brederode et al. 1991) and in the ventrolateral secondary somatosensory cortex, SmII (Wise and Donaghue 1988). Mean number of PV+ neurons and standard error (x ± SEM) were calculated from counts done in the dorsomedial (DM) and ventrolateral (VL) regions of the somatosensory cortex either in: supragranular (I-II), granular (IV) and infragranular (V-VI) layers of each group (P18, P24, P30, P120, EC24 and EP18-30). Statistical evaluation of our results was performed using software SigmaStat 2.0. Histograms were generated with the data collected. Differences were significant at P< 0.05.


PV+ immunoreactive neurons were intensely stained throughout the perikaryon and cell processes in all experimental animals. The neurons were located in layers II through VI of the somatosensory cortex (Fig. 1 ). PV+ cells were bipolar or multipolar. Most neurons had their major axis perpendicularly oriented with respect to the cortical surface (Fig. 2). However, some bipolar cells of layer II and VI were horizontally oriented to the cortical surface. A dark band which includes PV immunoreactive fibers and terminal processes was visible in layer IV in non counterstained sections (Fig.1).

Fig. 1. Photomicrograph of the rat somatosensory cortex in postnatal day 30, showing parvalbumin (PV) immunoreactivity. Cortical layers are shown to the right. Note the uneven distribution of PV+ neurons and the neuropile band of immunoreactivity in layer IV (arrow heads). Scale bar = 160 µm.

Fig. 2. Photomicrograph of PV+ neurons found in infragranular layers of the rat somatosensory cortex in postnatal day 30. Note the intense immunoreactivity in cell bodies and cell processes. Major axis of PV+ neurons are arranged in different angles with respect to the cortical surface (top of the figure). Arrow heads = blood vessels. Scale bar = 30 µm.

The total number of PV+ immunoreactive neurons reached a peak on day 24 (6182) and gradually decreased by day 120 (4794, see Fig.3). A consistent difference between infragranular and supragranular layers was observed in all control groups (P18 to P120) either in the dorsomedial or ventrolateral areas. Infragranular layers had approximately 55% more PV+ neurons than supragranular layers (Fig.3). In P24, P30 and P120 a difference in the number of labeled neurons between dorsomedial and ventrolateral regions of the somatosensory cortex was observed. Dorsomedial regions had approximately 16% more PV+ neurons. This difference was statistically significant in all layers of the cerebral cortex of P30 (see Fig. 3). Finally, a posterior to anterior difference in the number of PV+ neurons was also detected. The latter disappears by P120.

Fig. 3. Mean number of PV+ neurons per 240 µm wide bands found in cortical supragranular (II-III), granular (IV) and infragranular (V-VI) layers during the postnatal development. Neurons of each experimental group have been summed for each laminae represented. DM= dorsomedial and VL= ventrolateral aspects of the somatosensory cortex at 18th, 24th, 30th and 120th postnatal days. * P< 0.05; ** P< 0.001 (Student test); n = number of PV+ neurons.

Animals subjected to enriched condition from day 3 to day 24 (EC24) show a reduction in the total number of PV+ neurons (4589) compare with P24 (6182; see Figs. 3 and 4). The decrement of immunoreactivity, approximately 26%, is noteworthy in the supragranular and infragranular layers in both dorsomedial and ventrolateral regions of the somatosensory cortex (Fig. 5). The dorsomedial-ventrolateral, the infragranular-supranular and the posterior-anterior differences in the number of PV+ neurons observed in P24 were retained in EC24 (Table I).

Fig. 4. Mean number of PV+ neurons per 240 µm wide bands found in supragranular (II-III), granular (IV) and infragranular (V-VI) layers. Neurons of each experimental condition have been summed for each laminae represented. DM= dorsomedial and VL= ventrolateral aspects of the somatosensory cortex in enriched condition (EC24) and enriched condition-persistence (EP18-30) groups. * P< 0.05; ** P< 0.001 (Student test); n = number of PV+ neurons.

Animals maintained under the stimulated condition until day 18 and sacrified on day 30 (EP18-30) show a reduction in the total number of PV+ neurons (4199) when compare with control animals P30 (5951; see Figs. 3 and 4). The reduction of PV immunoreactivity, approximately 29%, was detected in all cortical layers in both the dorsomedial and the ventrolateral regions of the somatosensory cortex (Fig. 5 and Table 1). It is important to point out that the differences in the number of PV+ neurons observed in the three dimension of the cerebral cortex (infragranular-supragranular, dorsomedial-ventrolateral and posterior-anterior) in P30 persist inEP18-30.

Table 1. Effect of environmental enrichment upon the number of PV+ neurons.    

  Values are numbers of PV+ neurons per cortical layer in dorsomedial (DM) and ventrolateral (VL) regions of the somatosensory cortex, in controls (P24 and P30) and enriched condition (EC24 and EP18-30) animals. EC24 and EP18-30 show a decrement in percentage of PV+ neurons in comparison with their respective control groups.
  P24 EC24 P30 EP18-30  
Layer II-III 1056 740 1014 702  
DM   30%¯   31%¯  
Layer II-III 7330 529 7530 335  
VL   28%¯   56%  
Layer V-VI 1728 1224 1666 1372  
DM   29%¯   18%¯  
Layer V-VI 1587 1136 1456 1008  
VL   28%¯   31%¯  

Fig. 5. Dorsomedial (DM) and ventrolateral (VL) regions of the somatosensory cortex were compared in control P24 v/s enriched condition EC24 groups (left panel) and control P30 v/s enriched condition-persistence EP18-30 groups (right panel). Comparison between mean number of PV+ neurons in supragranular (II-III) and infragranular (V-VI) layers in dorsomedial DM and ventrolateral VL aspects of the neocortex of the rat are depicted. *P< 0,002; **P< 0,001.


Our immunohistochemical study in the somatosensory cortex of albino rats shows that the definitive distribution patterns of PV+ neurons is established between postnatal days 24 and 30. Distribution of PV+ neurons in the somatosensory cortex follows a peculiar pattern. More PV+ neurons were found in infragranular than supragranular, in dorsomedial than ventrolateral and in posterior than anterior regions of the cerebral cortex. It is important to point out that the onset of PV expression appears by postnatal day 8 in the infragranular layers and three days later in the supragranular layers of the neocortex (Alcantara et al., 1993). Likewise, PV+ neurons are first seen in SmI and two days later in SmII. Moreover, PV expression appears earlier in occipital and parietal cortices than in frontal regions of the neocortex (Alcantara et al., 1993). It is noteworthy that both medio-lateral and posterior-anterior PV expression patterns are reverse with respect to cortical histogenesis and differentiation (Smart, 1983; Miller, 1988). It should be noted that the development of PV immunoreactivity in the cerebral cortex correlates "with the maturation of different cortical-related functions: affective and localizing response to painful stimuli, suckling, olfaction, vision, hearing, motor coordination and finally integrative behavior" (Alcantara et al., 1993). This probably explains the peculiar order of PV expression not strictly linked to developmental process as occurs with the synthase of nitric oxide NADPH-d (Bravo et al., 1997).

Environmental enrichment from postnatal day 3 to 24 the critical period of developmentinduces, as shown in our experiment EC24, a significant reduction of PV+ neurons. Density changes affect supragranular and infragranular layers, although the tridimensional pattern of distribution detected in control animals is retained (Figs. 3 and 4). Using the same experimental paradigm in two independent studies, we have found that nicotinamide adenine dinucleotide phosphate diaphorase (NADPH)+ neurons are reduced about 10% (Fernández et al., 1998) v/s 26% (this study, Fig. 5) in PV+ neurons . This difference could be explained by the fact that the onset of PV expression starts and develops during the critical period of development, a stage in which the experimental animals are already under the effects of polysensorial stimulation, while NADPH expression starts prenatally (Bredt & Snyder 1994; Soriano et al., 1992) . In both cases, NADPH and PV, distribution patterns are retained (Bravo et al., 1997; Fernández et al., 1998). These peculiar patterns suggest some degree of stability in the distribution of specific population of nonpyramidal neurons of the cerebral cortex. In the same line, a recent study by our group using the Golgi-Cox-Sholl technique (Fernández et al., 1997) shows that environmental enrichment, during the critical period of development, induces a significant increment of dendritic arborizations in the pyramidals neurons of layer V in the visual cortex. This feature shows that neural plasticity involves quantitative changes such as cell reduction with an increasing soma size and dendritic expansion, in response to environmental manipulation (Diamond et al., 1964; Greenough & Volkman 1973; Fernández et al., 1997). Preliminary results obtained in our laboratory show that PV+ somatas of enriched condition animals EC24 are 6% larger in area than PV+ neurons of controls animals P24 (unpublished observations). Morphometric studies using different methods have demonstrated that environmental enrichment, training in a motor task and nutritional rehabilitation induce decreased neuronal densities associated with increments in dendritic arborization (Carughi et al., 1989; Venable et al., 1989; Diaz et al., 1994). The same explanation applies for the reduction of PV+ neurons, due to environmental enrichment. Nevertheless, other mechanisms, such as apoptosis, should be considered.

The magnitude of neuronal reduction induced by environmental enrichment in our study (26% in EC24 and 29% in EP18-30 experiments) is three times greater than that found in the neocortex of older rats (three months of age) submitted to enriched condition, for a longer period, from days 30 to 90 (Katz & Davies 1984). This situation could be explained in two complementary ways: the PV+ neurons are more sensitive to environmental manipulations, or the effects of environmental enrichment are age-dependent. The latter fits with the concept of critical period of development since, the earlier the onset of environmental enrichment the greater the neuronal changes. In this respect, a previous behavioral study (Venable et al.. 1988), using maze learning, showed the greater efficacy of preweaning than postweaning environmental enrichment in problem-solving ability in rats.

The effects of the enriched environments seem to persist in time (at least twelve days) since EP18-30 animals, present a similar reduction on PV+ neurons (29%) with respect to controls P30 (Fig. 5). The gradients of distribution observed in PV immunoreactive neurons in EC24 pups are consistent with the pattern found in EP18-30 pups (fig.4). In this respect, Venable et al. (1988) showed that preweaning enrichment from day 10 to 24, produce long term effects in problem-solving ability in rats tested on day 100, result supported by our EP18-30 experiments.

PV expression in the neocortex of the rat is a postnatal process that extends from day 8 to 21 (Alcantara et al., 1993), and runs in parallel with the critical period of cortical development (Venable et al. 1989). In addition, PV+ neurons seem to be, according to our results, substantially more sensitive than NADPH-d+ neurons to environmental enrichment. These facts support the notion that PV immunoreactivity is a useful marker to explore the effects of environmental manipulation during the critical period of development in the cerebral cortex.


The authors are grateful to Drs. Jaime Olavarría, Fernando Torrealba, Samuel Ruiz and Pablo Caviedes for critical reading and helpful comments on the manuscript. We also thanks Miguel Sanhueza for technical assistance. 

INZUNZA, O.; BRAVO, H. & FERNÁNDEZ, V. El enriquecimiento ambiental durante el desarrollo postnatal temprano disminuye la expresión de parvalbúmina en la corteza somatosensorial de la rata Int. J. Morphol., 21(3):181-189, 2003.

RESUMEN: La expresión de parvalbúmina (PV) en el cerebro de la rata comienza en el día postnatal 8, comprometiendo una población heterogenea de neuronas GABAérgicas no piramidales. En el presente trabajo hemos realizado un estudio utilizando inmunohistoquímica en animals controles y experimentales sometidos a un ambiente enriquecido con estimulación polisensorial entre los dias 3 y 18 o entre el 3 y el 24. El recuento de las neuronas PV+ fue realizado en las regiones dorsomediales y ventrolaterales de la corteza somatosensorial. En los animales control las neuronas PV+ alcanzan un máximo en el día 24, disminuyendo hacia el día 120. En estos animales se encontró un patrón peculiar en el cual las neuronas inmunoreactivas son más numerosas en la región dorsomedial que en la región ventrolateral así como más numerosas en las capas infragranulares que en las supragranulares y en las zonas posteriores que en las anteriores de la corteza cerebral. Las diferencias observadas en estas tres dimensiones estan bien definidas en el día 24. Ratas expuestas al ambiente enriquecido desde el día 3 al 24 muestran una reducción del 26% en el número de neuronas PV+ . El efecto del enriquecimiento ambiental persiste al menos 12 días dado que animale s sometidos a estas condiciones desde el día 3 al 18 y sacrificados el día 30 presentan también una reducción similar (29%).

El enriquecimiento ambiental polisensorial induce una significativa reducción de las neuronas inmunoreactivas a parvalbúmina, manteniendo sin embargo el patrón de distribución general expresado anteriomente. Estos hallazgos indican un grado de estabilidad en la expresión de parvalbúmina en la corteza somatosensorial de la rata.

PALABRAS CLAVE: 1. Parvalbumina; 2. Corteza cerebral; 3. Estimulación polisensorial; 4. Rata. 5. Inmunohistoquímica.


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Correspondence to:

Prof. Dr. Oscar Inzunza
Departamento de Anatomía
Escuela de Medicina
Pontificia Universidad Católica de Chile
Casilla 114-D

E. mail: oinzunza@

Received : 28-04-2003
Accepted : 25-05-2003 

* Departamento de Anatomía, Escuela de Medicina, Pontificia Universidad Católica de Chile, Chile.
** Instituto de Ciencias Biomédicas, Programa de Fisiología y Biofísica, Facultad de Medicina, Universidad de Chile, Chile.

This work was supported by FONDECYT Grant 1950649.

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