Psicothema, 2000. Vol. Vol. 12 (nº 2). 293-295

Luis M. García-Moreno, José M. Cimadevilla^*, Héctor González-Pardo^* y Jorge L. Arias^*

Recientemente se han encontrado diferencias en cuanto a la actividad neural de la formación hipocámpica en sus zonas dorsal y ventral. Estas diferencias podrían jugar un importante papel en cuanto a la implicación diferencial de estas zonas en el aprendizaje y la memoria. Aplicamos aquí la técnica histológica de impregnación argéntica de las regiones organizadoras del nucleolo (AgNORs) con el fin de analizar la actividad biosintética de las areas hipocampales CA1 y CA3 en las zonas dorsal y ventral de ratas Wistar. Se cuantificaron el número y área media de los AgNORs presentes en sus neuronas, que son indicadores indirectos de la actividad de síntesis proteica. Los resultados muestran una actividad biosintética significativamente mayor en la región dorsal de ambas áreas estudiadas, que concuerda con estudios anteriores y se discuten sus implicaciones funcionales.

Diferencias funcionales entre el hipocampo dorsal y ventral reveladas con la tinción AgNOR. Recently differences were reported concerning the dorsal and ventral zones of hippocampal formation. These differences may play an important role regarding the differential involvement of both zones in learning and memory. We use here the silver staining of nucleolar organizer regions (AgNORs) histological technique in order to analyze the biosynthetic activity of CA1 and CA3 hippocampal areas in dorsal and ventral zones from Wistar rats. We quantified the mean number and area of AgNORs in their neurons, which are considered as indirect indexes of protein synthesis activity. The results show a significantly higher biosynthetic activity in the dorsal region from the areas studied, which agrees with previous research. Functional implications derived from our results are discussed in this paper.

Since the first descriptions of the famous case of patient H.M. by Scoville and Milner (1957), the important role of the hippocampus in the formation of memory has been extensively documented. Until now, a vast majority of actual experimental work with animals and humans has been done considering the hippocampus as a homogeneous structure with a unified functional role (Eichenbaum et al., 1999; O’Keefe et al., 1998). However, numerous studies report the morphological, neurochemical and functional differences between dorsal and ventral zones from hippocampus (Moser et al., 1993; Jung et al., 1994; Bannerman et al., 1999). According to these findings, the experimental research made on the hippocampus, mainly by lesion techniques, shows that the effect of such disruption is different depending on the affected site (Moser et al., 1993; Lorenzini et al., 1997; Levin et al., 1999). Additionally, the behavioral effects are supported by other neurochemical and physiological heterogeneity (Bruinink and Bischoff, 1993; Jung et al., 1994).

Although a number of techniques offer a good approach to the functional study of central nervous system structures, silver staining of nucleolar organizer regions (AgNORs) is considered a rapid and easy way to obtain an estimation of protein synthesis rate (Crocker, 1996). This technique makes it possible to label the fibrillar centers, a nucleolar component involved in the synthesis of ribosomal RNA. The fibrillar centers are related morphologically with the NORs, sites composed of RNA and proteins, many of them argyrophilic. By using this technique these NOR-associated proteins are selectively stained; the result of this reaction is known as AgNOR dots, and the number and area of AgNORs are an accurate index of activity and cell proliferation in terms of protein synthesis (Derenzini et al., 1992; Ploton et al., 1992). The AgNOR technique alone or combinations with other histological stains have been used in various studies of the CNS with promising results in animals and humans (Fushiki et al., 1995; González-González et al., 1996; García-Moreno et al., 1997).

By taking into consideration these studies, the present work aimed to evaluate the biosynthetic activity in CA1 and CA3 hippocampal areas from both the dorsal and ventral zones. The above-mentioned AgNOR parameters were selected as adequate indexes of neural transcriptional activity.

Materials and method

Eight male adult Wistar rats (120 days of age) from the University of Oviedo vivarium were used. They were housed at a constant temperature of 21±1 ºC and a 12-hour light/dark period (lights on from 8:00 to 20:00 hours) with water and food available ad libitum. All animals were handled following strictly the European directives for care and use of laboratory animals (EC 86/609).

Rats were anesthetized with ether and transcardially perfused with 10% formaldehyde in phosphate buffer (0.1M, pH 7.4). Their brains were carefully removed and maintained in the same fixative for several days. Later, a tissue block comprising the region between the optic chiasm and posterior hypothalamus was embedded in paraffin. The blocks were sectioned in 15 µm-thick coronal sections, selecting 2 out of 12 sections across the hippocampus. From each pair, only one was selected for AgNOR stain according to Ploton et al. (1992) method, with a total of 11-15 equidistant sections obtained from the entire hippocampus.

The AgNOR parameters chosen were the mean AgNOR number and area per neuron, since it was successfully used in other clinical and experimental studies (García-Moreno et al., 1993; Crocker, 1996; González-González et al., 1996); it seems that the higher the mean AgNOR number and area, the higher the transcriptional activity is. By using an optical microscope (Leica series, Germany) and a computer image analysis system (Leica Q-Win) a total of 100 pyramidal neurons were sampled from each animal in order to measure their AgNOR dots. The lateral geniculate nucleus was used as a reference to mark the boundary between the dorsal and ventral zones of hippocampus in its most caudal part.

Results

In order to analyze the differences between the dorsal and ventral hippocampus and the regions selected, a two-way (zone x region) repeated measures ANOVA was applied regarding the AgNOR parameters evaluated: mean AgNOR area and mean AgNOR number per neuron.

The results for the mean AgNOR number show an statistically significant difference between dorsal and ventral hippocampal zones (F _1,7 =17.5; P<0.01), as shown in figure 1. No significant differences were observed for region and its interaction with zone factor. In the case of the mean AgNOR area, no differences between dorsal and ventral zones in CA1 and CA3 were found (Fig. 3). However, significant differences were found for the region factor (F _1,7 =18.6; P<0.01), since mean values for CA3 were significantly higher than those from CA1 region for the latter AgNOR parameter.

Discussion

Our results show that there is a significant difference in the biosynthetic activity from dorsal and ventral regions from both CA1 and CA3 hippocampal areas. Moreover, the activity is higher in the dorsal region in basal condition. The parcellation of hippocampus into dorsal and ventral zones has been considered by other authors, which found morphological and functional differences that could explain the reported results (Moser et al., 1993; Jung et al., 1994). Thus, from a behavioral point of view, whereas dorsal hippocampal lesions facilitate active avoidance response in the shuttle-box, this is not the case for the ventral zone (Lorenzini et al., 1997). Conversely, after dorsal hippocampal lesions an impairment of spatial task performance is observed, not affected by ventral damage (Moser et al., 1993). Recent evidence supports a functional double dissociation between dorsal and ventral hippocampus related with different behaviors involving spatial learning and motor activity or contextually conditioned freezing (Bannerman et al., 1999; Richmond et al. 1999).

By using the silver staining of NORs, many differences were reported in other CNS structures according to their activity levels (Lafarga et al., 1995; Gonzalez-Gonzalez et al., 1996). Changes in neuronal transcriptional activity may be related in adult CNS with protein synthesis, and the latter would reveal the modification of several functional parameters such as long-term potentiation (LTP), since blocking biosynthetic activity ceases (Stanton and Sarvey, 1984; Otani et al., 1992). Additionally, the relationship between transcriptional activity and cell firing rate is well known, a fact that would explain the reported differences in the frequency and duration of electrical responses between dorsal and ventral hippocampus (Jung et al., 1994). We should take into consideration that the dorsal hippocampus receives the majority of sensorial input from the cortex and parahippocampal structures (Room and Groenewegen, 1986; Witter, Van Hoesen and Amaral, 1989); this would explain a higher synaptic activity of the dorsal subdivision, reflected in a higher biosynthetic activity.

The results concerning the higher activity of CA3 region, manifested by a high AgNOR area should be considered in synaptic terms. Particularly, the CA3 region receives a number of inputs by the mossy fibers from the dentate gyrus, and the latter from the entorhinal cortex via the perforant path; this means that all the information that the hippocampal formation receives establishes synaptic contacts mainly with CA3 and not CA1, so that the postsynaptic activity of CA3 pyramidal cell would be logically high and therefore its biosynthetic demands.

In conclusion, although in numerous investigations the hippocampus is considered as a whole, our data question this assumption clearly. Thus, it may be inappropriate to formulate hypotheses about a unitary function of hippocampal function. In this way, this work demonstrates that significant differences in activity are found between the dorsal and ventral hippocampus. By taking this results into consideration, future research is necessary in order to explain more precisely the behavioral and physiological significance of this heterogeneity in the hippocampal formation.

Acknowledgements

This work was supported by grants DEGS PB96-0318 from Ministerio de Educación y Cultura (Spain), FICYT (PBSAL98-08) from Principado de Asturias (Spain), and UCM PR64/99-8499.

Bannerman, D.M., Yee, B.K., Good, M.A., Heupel, M.J., Iversen, S.D. and Rawlins, J.N.P. (1999) Double dissociation of function within the hippocampus: a comparison of dorsal, ventral, and complete hippocampal cytotoxic lesions. Behavioral Neuroscience 113(6): 1170-1188.

Bruinink, A. and Bishoff, S. (1993) Dopamine D2 receptors are unevenly distributed in the rat hippocampus and are modulated differently than in striatum. European Journal of Pharmacology 245(2):157-164.

Crocker, J. (1996) Correlation between NOR sizes and numbers in non-Hodgkin’s lymphomas. Journal of Clinical Pathology 49: 8-11.

Derenzini, M., Farabegoli, F. and Treré, D. (1992) Relationship between interphase AgNOR distribution and nucleolar size in cancer cells. Journal of Histochemistry 24: 951-956.

Eichenbaum, H., Dudchenko, P., Wood, E., Shapiro, M. and Tanila, H. (1999) The hippocampus, memory, and place cells: is it spatial memory or a memory space? Neuron 23(2): 209-226.

Fushiki, S., Kinoshita, C., Tsutsumi, Y. and Nishizawa, Y. (1995) A combined staining method for argyrophilic nucleolar organizer regions and for glial fibrillary acidic protein in astrocytes of human brain. Acta Histochemica and Cytochemica 28(6): 533-538.

García-Moreno, L.M., Cimadevilla, J.M., González-Pardo, H., Zahonero, M.C. and Arias, J.L. (1997) NOR activity in hippocampal areas during the postnatal development and ageing. Mechanisms of Ageing and Development 97:173-181.

García-Moreno, L.M., Santín, L.J., Rubio, S., González-Pardo, H. and Arias, J.L. (1993) Effects of ethanol and diazepam on AgNOR neuronal activity in the medial mammillary nucleus. Psicothema 5(1): 125-134.

González-González, S., Díaz, F., Vallejo, G. and Arias, J.L. (1996) Functional sexual differences in rat mammillary bodies: a quantitative Ag-NOR study. Brain Research 736:1-6.

Jung, M.W., Wiener, S.I. and McNaughton, B.L. (1994) Comparison of spatial firing characteristics of units in dorsal and ventral hippocampus of the rat. Journal of Neuroscience 14(12): 7347-7356.

Lafarga, M., Andrés, M.A., Fernández Viadero, C., Villegas, J. and Berciano, M.T. (1995) Number of nucleoli and coiled bodies and distribution of fibrillar centres in differentiating Purkinje neurons of chick and rat cerebellum. Anatomy and Embriology 191(4): 359-367.

Levin, E.D., Channelle Christopher, N., Weaver, T., Moore, J. And Brucato, F. (1999) Ventral hippocampal ibotenic acid lesions block chronic nicotine-induced spatial working memory improvement in rats. Cognitive Brain Research 7: 405-410.

Lorenzini, C.G.A., Baldi, E., Bucherelli, C., Sacchetti, B and Tassoni, G. (1997) Role of ventral hippocampus in acquisition, consolidation and retrieval of rat’s passive avoidance response memory trace. Brain Research 768: 242-248.

Moser, E., Moser, M.B. and Andersen, P. (1993) Spatial learning impairment parallels the magnitude of dorsal hippocampal lesions, but is hardly present following ventral lesions. Journal of Neuroscience 13(9):3916-3925.

O’Keefe, J., Burgess, N., Donnett, J.G., Jeffery, K.J. and Maguire, E.A. (1998) Place cells, navigational accuracy, and the human hippocampus. Philosophical Transactions from the Royal Society of London B, 353(1373): 1333-1340.

Otani, S., Roisin-Lallemand, M.P. and Ben-Ari, Y. (1992) Enhancement of extracellular protein concentrations during long-term potentiation in the rat hippocampal slice. Neuroscience 47(2): 265-272.

Ploton, D., Visseaux-Coletto, B., Canellas, J.C., Bourzat, C., Adnet, J.J., Lechki, C. and Bonnet, N. (1992) Semiautomatic quantification of silver-stained nucleolar organizer regions in tissue sections and cellular smears. Analytical and Quantitative Cytology and Histology 14(1):14-22.

Richmond, M.A., Yee, B.K., Pouzet, B., Veenman, L., Rawlins, J.N.P., Feldon, J. and Bannerman, D.M. (1999) Dissociating context and space within the hippocampus: effects of complete,dorsal, and ventral excitotoxic hippocampal lesions on conditioned freezing and spatial learning. Behavioral Neuroscience 113(6): 1189-1203.

Room, P. and Groenewegen, H.J. (1986) Connections of the parahippocampal cortex. I cortical afferents. Journal of Comparative Neurology 209: 69-78.

Scoville, W.B. and Milner, B. (1957) Loss of recent memory after bilateral hippocampal lesions. Journal of Neurology, Neurosurgery and Psychiatry 20: 11-2.

Stanton, P.K. and Sarvey, J.M. (1984) Blockade of long-term potentiation in rat hippocampal CA1 region by inhibitors of protein synthesis. Journal of Neuroscience 4(12): 3080-3088.

Witter, M.P., Van Hoesen, G.W. and Amaral, D.G. (1989) Topographical organisation of the entorhinal projection to the dentate gyrus of the monkey. Journal of Neuroscience 54: 5-9.