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Fast spiking cells in rat hippocampus (CA1 region) contain the calcium-binding protein parvalbumin
Brain Research, 416 (1987) 369-374
369
Elsevier BRE 22390
Fast spiking cells in rat hippocampus (CA 1 region) contain the calcium-binding protein parvalbumin Yasuo Kawaguchi 1, Hironobu Katsumaru l, Toshio Kosaka 1, Claus W. Heizmann 2 and Kiyoshi Hama 1 lNational Institute for Physiological Sciences. Myodaiji, Okazaki (Japan) and :Institute of Pharmacology and Biochemistry, University of Ziirich-lrchel, Ziirich (Switzerland)
(Accepted 28 April 1987) Key words': Fast spiking cell; Calcium-bindingprotein; Parvalbumin; y-Aminobutyric acid (GABA)ergic neuron; Non-pyramidal
cell; Hippocampus; Intracellular injection of Lucifer yellow; Immunohistochemistry
Fast spiking cells in the CA 1region of the rat hippocampus were revealed as 7-aminobutyric acid (GABA)ergic non-pyramidal cells containing the calcium-binding protein parvalbumin by intracellular injection of Lucifer yellow in vitro in combination with posternbedding parvalbumin immunohistochemistry Calcium is known to regulate a large number of intracellular processes. Intracellular calcium may be buffered by calcium-binding proteins 13. Parvalbumin (PV), one of them, is distributed in skeletal muscle, brain, kidney and testis 7,s. In the central nervous system of the rat PV exists in a subpopulation of y-aminobutyric acid (GABA)ergic neurons 4,5,7,s, which are considered electrically and metabolically more active than companion neurons 4. In the hippocampus, GABAergic neurons are morphologically nonpyramidal cells 2°'23. Among hippocampal non-pyramidal cells fast spiking cells 15 form a physiologically distinct subclass 1°,18'19. Fast spiking cells are able to fire at a high frequency and they show no adaptation of spike frequency with sustained depolarization l°'ls'lg. The spatial distribution of fast spiking cells 1° corresponds very well to that of PV-containing GABAergic neurons in the rat hippocampus 5's,12. From their electrophysiologicai characteristics and the correspondence between their spatial distributions, there is a possibility that fast spiking cells are PV-containing GABAergic neurons. In the present paper, by combining intracellular staining and PV immunohistochemistry we directly demonstrate that hippocampal fast spiking cells contain PV.
Male Wistar rats (100-180 g) were deeply anaesthetized with ether and decapitated. Hippocampal slices, 350-400 ~m thick, were cut and placed in oxygenated solution (32 °C) for 1 h. A single slice was then transferred to the recording chamber, submerged beneath a superfusing oxygenated medium (31-33 °C) consisting of (in mM): NaC1 116.4, KCI 5.4, CaC12 4.0, MgSO 4 1.3, N a H C O 3 26.2, NaHzPO 4 1.0, and glucose 11.0. Concentrations of calcium in this solution are somewhat higher than normal in order to stabilize the cells 2 because recordings from fast spiking cells were shorter and less stable than those from pyramidal cells 15'19. As stable recordings could be done in this condition, fast spiking non-pyramidal cells were easily distinguished from pyramidal cells according to spike shape and discharge pattern 1° (see below). In the stratum pyramidale of hippocampal CA~ region, fast spiking cells were identified on the following criteria3"l°'15,18,19: (1) the spike-width at one-half amplitude of the maximal action potential is less than 0.40 ms; (2) large current pulses (>1 nA) elicit sustained trains of spikes; (3) each action potential is followed by a brief but large afterhyperpolarization (Fig. 1A). After the physiological identification Luci-
Correspondence: Y. Kawaguchi, National Institute for Physiological Sciences, Myodaiji, Okazaki 444, Japan.
0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
370
lO ms
F"
20 ms
C
50 mV I
lO ms Fig. 1. Electrophysiological recordings of a fast spiking cell (A,B) and a pyramidal cell (C). After completing the injection of Lucifer yellow, the spike was still observed (B). Depolarizing current pulses (thin underlines, A: 1.0 nA, B: 1.0 nA, C: 1.2 nA) were used to evoke spike discharges.
fer yellow (LY) was injected into fast spiking cells by passing a 1- to 2-nA negative D C current for 1 - 2 min from an electrode filled with a 5% aqueous solution of L Y , C H (Aldrich) (100-150 Mfl) or for 10 min from an electrode filled with a 0.5% solution of L Y . C H (Sigma) in 0.5 M potassium acetate (80-120 Mff2). A control sample of neurons filled with L Y was obtained by injecting CA~ pyramidal cells (Fig. 1C). For PV immunohistochemistry, we selected cells whose action potential mechanism was still intact after completing the injection of LY (Fig. 1B).
After completion of the LY injection slices were fixed in 4% paraformaldehyde in 0.l M phosphate buffer for 30 min at room temperature and at 4 °C overnight. They were then washed in the same buffer and cut at 50/~m with a V i b r a t o m c The sections containing the soma of a dye-marked cell were photographed with a fluorescence microscope. After being dehydrated and embedded in E p o n - A r a l d i t e , the sections were serially cut at 1/~m and mounted on albumin-coated glass slides. The specificity of the anti-PV serum developed by Heizmann 5'7's was tested by Western blotting. No cross-reactivity was observed with other Ca-binding proteins such as calmodulin, S-100 proteins, oncomodulin or calbidins. Semi-thin sections containing the labeled soma were processed for postembedding PV immunohistochemistry 21 using the peroxidase-antiperoxidase (PAP) method 22. Sections were treated for 30 min with ethanolic sodium hydroxide, followed by 3 washes in absolute ethanol and two washes in distilled water. Thereafter sections were incubated with the sera in a moist chamber at room temperature in the following sequence: normal goat serum (20%) for 30 min. rinse in phosphate-buffered saline (PBS): 2 days in PV antiserum diluted l:3000, three 10-rain washes in PBS: 1 h in goat anti-rabbit IgG diluted 1:50: three 10-min washes in PBS: rabbit P A P diluted 1:100 for 1 h: two 10-min washes in PBS: 10-rain wash in Tris-buffered saline (TBS); 0.05% diaminobenzidine tetrahydrochloride containing 0.01% H~O, in 0.05 M Tris buffer; rmse in TBS and then in PBS. Sections were treated for 5 min with 0.03% OsO~, rinsed several times in distilled water, and mounted in glycerol gelatin. By this immunohistochemical procedure, it was ascertained that PV-positive cells in immersion-fixed hippocampal slices used in the present experiment have the similar distributions to those in hippocampus fixed in situ by intracardial perfusion: most PV-positive cells existed in the stratum pyramidale, the stratum or]ens and subgranular zone of the dentate gyrus. Two types of controls were carried out to test the specificity of the immunohistochemical reaction. Primary antisera were replaced by (1) a serum preimmune to the antiserum against PV or (2) PV antiserum pretreated with excess PV. For producing pretreated antisera, 3.4 nmol (40ktg) of PV was added to
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Fig. 3. I m m u n o h i s t o c h e m i c a l detection of parvalbumin on a semi-thin section o f the s o m a of a fast spiking celt filled with Lucifer yellow. A and B: a pair of photomicrographs by fluorescence (A) and Nomarksi (B) optics of the same section which contains the soma (1) of a fast spiking cell injected with LY in the stratum pyramidale. C: immunostaining by anti-PV serum of a section adjacent to that shown in A and B. Two PV-positive somata (1 and 2) as well as n u m e r o u s PV-positive puncta are observed. The I_Y-filled fast spiking cell is PV-positive (l). Bar = 1 0 u m .
373 1 ml of 1:3000 diluted anti PV serum and incubated for 2 days at 4 °C. Seven fast spiking cells were injected with LY in the stratum pyramidale of h i p p o c a m p a l C A 1 region. All of them exhibited morphological features of nonpyramidal cells l°'ls'19 (Fig. 2). They had much fewer dendritic spines than p y r a m i d a l cells, or none at all 17. In semi-thin sections, intense LY fluorescence was retained in LY-injected cells (Fig. 3A). In adjacent sections processed for PV immunohistochemistry, all kY-injected fast spiking cells were PV-positive (Fig. 3Cl). It is unlikely that this positive PV-immunostaining might be due to non-specific staining caused by the injection of LY, because PV-positive non-pyramidal cells were observed that had not been injected with LY (Fig. 3C2) and LY-injected pyramidal cells (6 cases) were PV-negative. In other adjacent sections treated with a p r e i m m u n e serum (5 cases) or PV antiserum p r e t r e a t e d with excess PV antigen (2 cases), no immunostaining was observed. The present findings indicate that h i p p o c a m p a l fast spiking cells contain the calcium-binding protein PV. This idea is s u p p o r t e d by the similar spatial distributions of fast spiking cells l° and PV-containing neuronsS'~'t2: both are concentrated in the stratum pyramidale and subgranular zone of the dentate gy-
1 Adams, P.R., Brown, D.A. and Constanti, A., Pharmacological inhibition of the M-current, J. Physiol. (London), 332 (1982) 223-262. 2 Alger, B.E., Characteristics of a slow hyperpolarizing synaptic potential in rat hippocampal pyramidal cells in vitro, J. Neurophysiol., 52 (1984) 892-910. 3 Ashwood, T.J., Lancaster, B. and Wheal, H.V., In vivo and in vitro studies on putative interneurones in the rat hippocampus: possible mediators of feed-forward inhibition, Brain Research, 293 (1984) 279-291. 4 Cello, M.R., Parvalbumin in most ?~-aminobutyric acidcontaining neurons of the rat cerebral cortex, Science, 231 (1986) 995-997. 5 Celio, M.R. and Heizmann, C.W., Calcium-binding protein parvalbumin as a neuronal marker, Nature (London), 293 (1981) 300-302. 6 Halliwell, J.V. and Adams, P.R., Voltage-clamp analysis of muscarinic excitation in hippocampal neurons, Brain Research, 250 (1982) 71-92. 7 Heizmann, C.W., Parvalbumin, an intracellular calciumbinding protein; distribution, properties and possible roles in mammalian cells, Experientia, 40 (1984) 910-921. 8 Heizmann, C.W. and Celio, M.R., Immunolocalization of parvalbumin. Methods Enzymol., 139 (1987) 552-570. 9 Hotson, J.R. and Prince, D.A., A calcium-activated hyperpolarization follows repetitive firing in hippocampal neu-
rus. F u r t h e r m o r e , it is very likely that the fast spiking cells examined in the present study are G A B A e r g i c , because PV-containing neurons that have been looked at are G A B A e r g i c in the central nervous system of rats 45'7'8'1z. Fast spiking cells displayed very little or no adaptation of spike frequency 1°,15,~s. A d a p t a t i o n of action potential discharge of hippocampal pyramidal cells may be largely regulated by two potassium currents~4: the calcium-activated potassium currents 9 and the M-current 6. The conductance of the former depends on intracellular free calcium concentration ~6, whereas the latter is presumably not calciumsensitive 1. Therefore, these observations suggest that PV may act as an intracellular calcium-buffer suppressing the elevation of calcium concentration during repetitive firing and blocking the adaptation due to calcium-activating potassium conductance in fast spiking cells 4, in which the M-current may be also lacking or suppressed.
W e are grateful to Prof. M. Ito and Dr. C.J. Wilson for comments on the manuscript. W e acknowledge the excellent technical assistance of Miss K. Ohishi and Mr. K. Isogai.
rons, J. Neurophysiol., 43 (1980)409-419. 10 Kawaguchi, Y. and Hama, K., Two sybtypes of non-pyramidal cells in rat hippocampal formation identified by intracellular recording and HRP injection, Brain Research, 411 (1987) 190-195. 11 Knowles, W.D. and Schwartzkroin, P.A., Local circuit synaptic interactions in hippocampal brain slices, J. Neurosci.. 1 (1981) 318-322. 12 Kosaka, T., Katsumaru, H.Y., Hama, K., Wu, J.-Y. and Heizmann, C.W., GABAergic neuron containing the cae+-binding protein parvalbumin in the rat hippocampus and dentate gyrus, Brain Research, in press. 13 Kretsinger, R.H., Structure and evolution of calcium-modulated proteins, C.R.C. Crit. Rev. Biochem., 8 (1980) 119-174. 14 Madison, D.V. and Nicoll, R.A., Control of the repetitive discharge of rat CA1 pyramidal neurones in vitro, J, Physiol. (London), 354 (1984) 319-331. 15 McCormick, D.A., Connors, B.W., Lighthall, J.W. and Prince, D.A., Comparative electrophysiology of pyramidal and sparsely spiny stellate neurons of the neocortex, J. Neurophysiol., 54 (1985) 782-806. 16 Meech, R.W., Calcium-dependent potassium activation in nervous tissues, Rev. Biophys. Bioeng., 7 (1978) 1-18. 17 Peters, A. and Jones, E.G., Classification of cortical neurons. In E.G. Jones and A. Peters (Eds.), Cerebral Cortex,
374
Vol. 1; Cellular Components of the Cerebral Cortex, Plenum, New York, 1984, pp. 107-121. 18 Schwartzkroin, P.A. and Kunkel, D.D., Morphology of identified interneurons in the CA1 regions of guinea pig hippocampus, J. Comp. Neurol., 232 (1985)205-218. 19 Schwartzkroin, P.A. and Mathers, L.H., Physiological and morphological identification of a nonpyramidal hippocampal cell type, Brain Research, 157 (1978) 1-10. 20 Seress, L. and Ribak, C.E., GABAergic cells in the dentate gyrus appear to be local circuit and projection neurons, Exp. Brain Res., 50 (1983) 173-182.
21 Somogyi, P., Hodgson, A.J., Chubt~, l.W., Penke, B. and Erdei, A., Antisera to y-aminobutyric acid. II. Immunocytochemical application to the central nervous system, J. Histochem. Cytochem., 33 (1985) 240-248. 22 Sternberger, L.A., lmmunocytochemL~try. 2nd: edn., Wiley, New York, 1979. 23 Storm-Mathisen, J. and Ottersen, O P . . Neurotransmitters in the hippocampal formation. In F. Reinoso-Su~irez and C, Ajmone-Marsan (Eds.), (~rtical lmegration, Raven, New York, 1984. pp. 105-130.
369
Elsevier BRE 22390
Fast spiking cells in rat hippocampus (CA 1 region) contain the calcium-binding protein parvalbumin Yasuo Kawaguchi 1, Hironobu Katsumaru l, Toshio Kosaka 1, Claus W. Heizmann 2 and Kiyoshi Hama 1 lNational Institute for Physiological Sciences. Myodaiji, Okazaki (Japan) and :Institute of Pharmacology and Biochemistry, University of Ziirich-lrchel, Ziirich (Switzerland)
(Accepted 28 April 1987) Key words': Fast spiking cell; Calcium-bindingprotein; Parvalbumin; y-Aminobutyric acid (GABA)ergic neuron; Non-pyramidal
cell; Hippocampus; Intracellular injection of Lucifer yellow; Immunohistochemistry
Fast spiking cells in the CA 1region of the rat hippocampus were revealed as 7-aminobutyric acid (GABA)ergic non-pyramidal cells containing the calcium-binding protein parvalbumin by intracellular injection of Lucifer yellow in vitro in combination with posternbedding parvalbumin immunohistochemistry Calcium is known to regulate a large number of intracellular processes. Intracellular calcium may be buffered by calcium-binding proteins 13. Parvalbumin (PV), one of them, is distributed in skeletal muscle, brain, kidney and testis 7,s. In the central nervous system of the rat PV exists in a subpopulation of y-aminobutyric acid (GABA)ergic neurons 4,5,7,s, which are considered electrically and metabolically more active than companion neurons 4. In the hippocampus, GABAergic neurons are morphologically nonpyramidal cells 2°'23. Among hippocampal non-pyramidal cells fast spiking cells 15 form a physiologically distinct subclass 1°,18'19. Fast spiking cells are able to fire at a high frequency and they show no adaptation of spike frequency with sustained depolarization l°'ls'lg. The spatial distribution of fast spiking cells 1° corresponds very well to that of PV-containing GABAergic neurons in the rat hippocampus 5's,12. From their electrophysiologicai characteristics and the correspondence between their spatial distributions, there is a possibility that fast spiking cells are PV-containing GABAergic neurons. In the present paper, by combining intracellular staining and PV immunohistochemistry we directly demonstrate that hippocampal fast spiking cells contain PV.
Male Wistar rats (100-180 g) were deeply anaesthetized with ether and decapitated. Hippocampal slices, 350-400 ~m thick, were cut and placed in oxygenated solution (32 °C) for 1 h. A single slice was then transferred to the recording chamber, submerged beneath a superfusing oxygenated medium (31-33 °C) consisting of (in mM): NaC1 116.4, KCI 5.4, CaC12 4.0, MgSO 4 1.3, N a H C O 3 26.2, NaHzPO 4 1.0, and glucose 11.0. Concentrations of calcium in this solution are somewhat higher than normal in order to stabilize the cells 2 because recordings from fast spiking cells were shorter and less stable than those from pyramidal cells 15'19. As stable recordings could be done in this condition, fast spiking non-pyramidal cells were easily distinguished from pyramidal cells according to spike shape and discharge pattern 1° (see below). In the stratum pyramidale of hippocampal CA~ region, fast spiking cells were identified on the following criteria3"l°'15,18,19: (1) the spike-width at one-half amplitude of the maximal action potential is less than 0.40 ms; (2) large current pulses (>1 nA) elicit sustained trains of spikes; (3) each action potential is followed by a brief but large afterhyperpolarization (Fig. 1A). After the physiological identification Luci-
Correspondence: Y. Kawaguchi, National Institute for Physiological Sciences, Myodaiji, Okazaki 444, Japan.
0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
370
lO ms
F"
20 ms
C
50 mV I
lO ms Fig. 1. Electrophysiological recordings of a fast spiking cell (A,B) and a pyramidal cell (C). After completing the injection of Lucifer yellow, the spike was still observed (B). Depolarizing current pulses (thin underlines, A: 1.0 nA, B: 1.0 nA, C: 1.2 nA) were used to evoke spike discharges.
fer yellow (LY) was injected into fast spiking cells by passing a 1- to 2-nA negative D C current for 1 - 2 min from an electrode filled with a 5% aqueous solution of L Y , C H (Aldrich) (100-150 Mfl) or for 10 min from an electrode filled with a 0.5% solution of L Y . C H (Sigma) in 0.5 M potassium acetate (80-120 Mff2). A control sample of neurons filled with L Y was obtained by injecting CA~ pyramidal cells (Fig. 1C). For PV immunohistochemistry, we selected cells whose action potential mechanism was still intact after completing the injection of LY (Fig. 1B).
After completion of the LY injection slices were fixed in 4% paraformaldehyde in 0.l M phosphate buffer for 30 min at room temperature and at 4 °C overnight. They were then washed in the same buffer and cut at 50/~m with a V i b r a t o m c The sections containing the soma of a dye-marked cell were photographed with a fluorescence microscope. After being dehydrated and embedded in E p o n - A r a l d i t e , the sections were serially cut at 1/~m and mounted on albumin-coated glass slides. The specificity of the anti-PV serum developed by Heizmann 5'7's was tested by Western blotting. No cross-reactivity was observed with other Ca-binding proteins such as calmodulin, S-100 proteins, oncomodulin or calbidins. Semi-thin sections containing the labeled soma were processed for postembedding PV immunohistochemistry 21 using the peroxidase-antiperoxidase (PAP) method 22. Sections were treated for 30 min with ethanolic sodium hydroxide, followed by 3 washes in absolute ethanol and two washes in distilled water. Thereafter sections were incubated with the sera in a moist chamber at room temperature in the following sequence: normal goat serum (20%) for 30 min. rinse in phosphate-buffered saline (PBS): 2 days in PV antiserum diluted l:3000, three 10-rain washes in PBS: 1 h in goat anti-rabbit IgG diluted 1:50: three 10-min washes in PBS: rabbit P A P diluted 1:100 for 1 h: two 10-min washes in PBS: 10-rain wash in Tris-buffered saline (TBS); 0.05% diaminobenzidine tetrahydrochloride containing 0.01% H~O, in 0.05 M Tris buffer; rmse in TBS and then in PBS. Sections were treated for 5 min with 0.03% OsO~, rinsed several times in distilled water, and mounted in glycerol gelatin. By this immunohistochemical procedure, it was ascertained that PV-positive cells in immersion-fixed hippocampal slices used in the present experiment have the similar distributions to those in hippocampus fixed in situ by intracardial perfusion: most PV-positive cells existed in the stratum pyramidale, the stratum or]ens and subgranular zone of the dentate gyrus. Two types of controls were carried out to test the specificity of the immunohistochemical reaction. Primary antisera were replaced by (1) a serum preimmune to the antiserum against PV or (2) PV antiserum pretreated with excess PV. For producing pretreated antisera, 3.4 nmol (40ktg) of PV was added to
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Fig. 3. I m m u n o h i s t o c h e m i c a l detection of parvalbumin on a semi-thin section o f the s o m a of a fast spiking celt filled with Lucifer yellow. A and B: a pair of photomicrographs by fluorescence (A) and Nomarksi (B) optics of the same section which contains the soma (1) of a fast spiking cell injected with LY in the stratum pyramidale. C: immunostaining by anti-PV serum of a section adjacent to that shown in A and B. Two PV-positive somata (1 and 2) as well as n u m e r o u s PV-positive puncta are observed. The I_Y-filled fast spiking cell is PV-positive (l). Bar = 1 0 u m .
373 1 ml of 1:3000 diluted anti PV serum and incubated for 2 days at 4 °C. Seven fast spiking cells were injected with LY in the stratum pyramidale of h i p p o c a m p a l C A 1 region. All of them exhibited morphological features of nonpyramidal cells l°'ls'19 (Fig. 2). They had much fewer dendritic spines than p y r a m i d a l cells, or none at all 17. In semi-thin sections, intense LY fluorescence was retained in LY-injected cells (Fig. 3A). In adjacent sections processed for PV immunohistochemistry, all kY-injected fast spiking cells were PV-positive (Fig. 3Cl). It is unlikely that this positive PV-immunostaining might be due to non-specific staining caused by the injection of LY, because PV-positive non-pyramidal cells were observed that had not been injected with LY (Fig. 3C2) and LY-injected pyramidal cells (6 cases) were PV-negative. In other adjacent sections treated with a p r e i m m u n e serum (5 cases) or PV antiserum p r e t r e a t e d with excess PV antigen (2 cases), no immunostaining was observed. The present findings indicate that h i p p o c a m p a l fast spiking cells contain the calcium-binding protein PV. This idea is s u p p o r t e d by the similar spatial distributions of fast spiking cells l° and PV-containing neuronsS'~'t2: both are concentrated in the stratum pyramidale and subgranular zone of the dentate gy-
1 Adams, P.R., Brown, D.A. and Constanti, A., Pharmacological inhibition of the M-current, J. Physiol. (London), 332 (1982) 223-262. 2 Alger, B.E., Characteristics of a slow hyperpolarizing synaptic potential in rat hippocampal pyramidal cells in vitro, J. Neurophysiol., 52 (1984) 892-910. 3 Ashwood, T.J., Lancaster, B. and Wheal, H.V., In vivo and in vitro studies on putative interneurones in the rat hippocampus: possible mediators of feed-forward inhibition, Brain Research, 293 (1984) 279-291. 4 Cello, M.R., Parvalbumin in most ?~-aminobutyric acidcontaining neurons of the rat cerebral cortex, Science, 231 (1986) 995-997. 5 Celio, M.R. and Heizmann, C.W., Calcium-binding protein parvalbumin as a neuronal marker, Nature (London), 293 (1981) 300-302. 6 Halliwell, J.V. and Adams, P.R., Voltage-clamp analysis of muscarinic excitation in hippocampal neurons, Brain Research, 250 (1982) 71-92. 7 Heizmann, C.W., Parvalbumin, an intracellular calciumbinding protein; distribution, properties and possible roles in mammalian cells, Experientia, 40 (1984) 910-921. 8 Heizmann, C.W. and Celio, M.R., Immunolocalization of parvalbumin. Methods Enzymol., 139 (1987) 552-570. 9 Hotson, J.R. and Prince, D.A., A calcium-activated hyperpolarization follows repetitive firing in hippocampal neu-
rus. F u r t h e r m o r e , it is very likely that the fast spiking cells examined in the present study are G A B A e r g i c , because PV-containing neurons that have been looked at are G A B A e r g i c in the central nervous system of rats 45'7'8'1z. Fast spiking cells displayed very little or no adaptation of spike frequency 1°,15,~s. A d a p t a t i o n of action potential discharge of hippocampal pyramidal cells may be largely regulated by two potassium currents~4: the calcium-activated potassium currents 9 and the M-current 6. The conductance of the former depends on intracellular free calcium concentration ~6, whereas the latter is presumably not calciumsensitive 1. Therefore, these observations suggest that PV may act as an intracellular calcium-buffer suppressing the elevation of calcium concentration during repetitive firing and blocking the adaptation due to calcium-activating potassium conductance in fast spiking cells 4, in which the M-current may be also lacking or suppressed.
W e are grateful to Prof. M. Ito and Dr. C.J. Wilson for comments on the manuscript. W e acknowledge the excellent technical assistance of Miss K. Ohishi and Mr. K. Isogai.
rons, J. Neurophysiol., 43 (1980)409-419. 10 Kawaguchi, Y. and Hama, K., Two sybtypes of non-pyramidal cells in rat hippocampal formation identified by intracellular recording and HRP injection, Brain Research, 411 (1987) 190-195. 11 Knowles, W.D. and Schwartzkroin, P.A., Local circuit synaptic interactions in hippocampal brain slices, J. Neurosci.. 1 (1981) 318-322. 12 Kosaka, T., Katsumaru, H.Y., Hama, K., Wu, J.-Y. and Heizmann, C.W., GABAergic neuron containing the cae+-binding protein parvalbumin in the rat hippocampus and dentate gyrus, Brain Research, in press. 13 Kretsinger, R.H., Structure and evolution of calcium-modulated proteins, C.R.C. Crit. Rev. Biochem., 8 (1980) 119-174. 14 Madison, D.V. and Nicoll, R.A., Control of the repetitive discharge of rat CA1 pyramidal neurones in vitro, J, Physiol. (London), 354 (1984) 319-331. 15 McCormick, D.A., Connors, B.W., Lighthall, J.W. and Prince, D.A., Comparative electrophysiology of pyramidal and sparsely spiny stellate neurons of the neocortex, J. Neurophysiol., 54 (1985) 782-806. 16 Meech, R.W., Calcium-dependent potassium activation in nervous tissues, Rev. Biophys. Bioeng., 7 (1978) 1-18. 17 Peters, A. and Jones, E.G., Classification of cortical neurons. In E.G. Jones and A. Peters (Eds.), Cerebral Cortex,
374
Vol. 1; Cellular Components of the Cerebral Cortex, Plenum, New York, 1984, pp. 107-121. 18 Schwartzkroin, P.A. and Kunkel, D.D., Morphology of identified interneurons in the CA1 regions of guinea pig hippocampus, J. Comp. Neurol., 232 (1985)205-218. 19 Schwartzkroin, P.A. and Mathers, L.H., Physiological and morphological identification of a nonpyramidal hippocampal cell type, Brain Research, 157 (1978) 1-10. 20 Seress, L. and Ribak, C.E., GABAergic cells in the dentate gyrus appear to be local circuit and projection neurons, Exp. Brain Res., 50 (1983) 173-182.
21 Somogyi, P., Hodgson, A.J., Chubt~, l.W., Penke, B. and Erdei, A., Antisera to y-aminobutyric acid. II. Immunocytochemical application to the central nervous system, J. Histochem. Cytochem., 33 (1985) 240-248. 22 Sternberger, L.A., lmmunocytochemL~try. 2nd: edn., Wiley, New York, 1979. 23 Storm-Mathisen, J. and Ottersen, O P . . Neurotransmitters in the hippocampal formation. In F. Reinoso-Su~irez and C, Ajmone-Marsan (Eds.), (~rtical lmegration, Raven, New York, 1984. pp. 105-130.