Histochemical localization of cytochrome oxidase in the hippocampus: Correlation with specific neuronal types and afferent pathways

Neuroscience

Vol. 7, No. 10, pp. 2337 to 2361, 1982

0306-4522/82/102337-25$03.00/O Pergamon Press Ltd 0 1982 IBRO

Printed in Great Britain

HISTOCHEMICAL LOCALIZATION OF CYTOCHROME OXIDASE IN THE HIPPOCAMPUS: CORRELATION WITH SPECIFIC NEURONAL TYPES AND AFFERENT PATHWAYS G. H. KAGEYAMA*and M. T. T. WONG-RILEY? Department of Anatomy, University of California, San Francisco, San Francisco, CA 94143 tDepartment of Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, U.S.A. Abstract-Cytochrome oxidase was histochemically localized in the hippocampus and dentate gyrus of various species of mammals. The most intense staining was observed within stratum moleculare of areas CAL-3 and the outer molecular layer of the dentate gyrus, as well as the somatic and basal dendritic layers of CA3. These regions correspond to the synaptic terminal fields of major excitatory afferent pathways to the hippocampus. The somata of CA3 pyramidal cells and various interneurons were more intensely stained than CA1 pyramidal cells and dentate granule ceils, and these levels appeared to correlate positively with their reported rates of spontaneous firing. 65 At the electron-microscopic levei, the highest con~ntrations of densely reactive mitochondria were localized within the distal apical dendritic profiles of principai cells (granule and pyramidal) and certain interneutons (pyramidal basket and stratum pyramidale interneurons). The specific layers in which these structures were found are known to receive intense excitatory input from the perforant pathway. High concentrations of reactive mitochondria were also observed within the somata and proximal dendrites of CA3 pyramidal cells and various interneurons, confirming our light-microscopic observations. These results demonstrated that not only can soma and dendrites of the same cell have disparate but distinct levels of cytochrome oxidase activity, but the pattern of reactivity within a neuron’s apical and basal dendrites, or even within specific dendritic segments of the same dendrite can be quite different. While the levels of somatic reactivity correlate with reported levels of spontaneous and/or synaptic activity, the degree of dendritic and somatic staining appeared to be more closely related to the intensity of convergent and/or pathway-specific excitatory synaptic input.

In recent years there has been a growing interest in the relationship between energy metabolism and neuronal activity in the central nervous system. Since these processes are tightly coupled,36 one would expect that normal and experimentally altered regional variations in neuronal activity would be reflected by corresponding differences in the levels of energy metabolism. Accordingly, it has been shown that functionally active areas in the normal brain have elevated levels of glucose utilizationso~sg and oxidative enzymatic activity.2~,70 It has also been demonstrated that immediate adjustments in the rate of 2-deoxyglucose uptake5’ and long-term adjustments in the levels of cytochrome oxidase activity (cytochrome c oxidase; ferrocytochrome c: oxygen oxidoreductase EC 1.9.3.1)7’~73~74~75~76 accompany experimentally-induced decreases or increases in the level of neuronal activity within specific sensory pathways. * Present address: Department of Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Road, P.O. Box 26509, Milwaukee, WI 53226, U.S.A. Abbreoiatians: CO, cytochrome oxidase; SP, Stratum pyramidale.

The distribution of cytochrome oxidase (CO) reactive neurons mainly within functionally-active regions of the brain such as the auditory system70.74 suggests that the intensity of CO staining within somata may be related to the frequency of a neuron’s spontaneous spiking or synaptically-evoked activity. However, oxidative enzymatic levels may not be evenly distributed among different parts of the same cell, since in many regions of the brain, CO staining within neuronal processes in the neuropil may differ from that in the cell bodies.Z0*70*71*74For example, in the mouse somatosensory barrel fields, relatively non-reactive somata located in the barrel walls presumably give rise to highly reactive dendrites that are located within the barrel centers.72.76 Moreover, zones of high CO activity often coincide with terminal fields of major afferent pathways, such as the barrel centers of somatosensory cortex and lamina IV of primary visual cortex.7’,76 This suggests that there may be some pathway specificity associated with the distribution of CO reactivity in the nervous system. Based on these findings, we hypothesize that the level of CO activity may vary between the soma and dendrites of the same cell, and that high levels of CO activity may be related to spontaneous spiking ac-

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G. l-1. Kageyama and M. T. T. Wang-Rile!

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tivity and/or to pathway-specific synaptic input. The hippocampus is selected for this study because it is ideally organized both structurally and functionally for testing our hypotheses. First, the somata and dendrites of the principat cells are clearly segregated into distinct laminae so that it can be determined whether or not CO activity may vary between these structures. Second, at least four histologically-identifiable cell types with known spontaneous firing rates have been reported6a.65 and can be compared with their level of CO staining. Lastly, the dendrites of the principal cells are oriented perpendicular to layers of pathwayspecific input.8,9,47 These regions can again be compared for their levels of CO activity.

spective fields. The distribution of CO activity tn the hippocampus and dentate gyrus of rat. rna~~~~~~l~, cat

and woodrat are shown in Figs 2%. In this report. the relative level of activity and staining are used interchangeably, although in both cases they could represent a relative amount of enzyme or level of enzyme activity per mitochondrion, or a relative number of mitochondria per unit volume. At the light-microscopic level. the distributio1~ of cytochrome oxidase staining in the hippocampus and dentate gyrus was remarkably similar in all species examined. However, a few minor variations were noted in different species and/or individual animals. For example,

EXPERIMENTAL

PROCEDURES

A total of 37 animals of 10 different mammalian species: 2 bats (Myotis), 8 albino mice, 4 albino (Sprague-Dawley) and 8 pigmented (Long Evans) rats, 2 woodrats, 2 Mongolian gerbils, 1 ground squirrel, 6 cats, 3 squirrel monkeys and I macaque monkey were used for this study. All animals were normal adults. Bats and rodent species were anesthetized with intraperitoneal (i.p.) injections of chloral hydrate (40 mg/lOG g body weight) while cats and monkeys were anesthetized with sodium pentobarbital i.p. (30 and 24mg/kg body weight, res~ctively). All animals were perfused through the left ventricle with an initial warm (37°C) buffered saline flush followed by a cold (4°C) mixture of 2.5% paraformaldehyde and l.S?< glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) with 4% sucrose. The brains were immediately removed and placed in cold fixative for 1-3 h depending on the size of the brain. Prolonged fixation time tends to reduce CO activity (our personal observations: see also Hanker’*). All brains were rinsed in three changes of original buffer. Those brains used for light-microscopic studies were cryoprotected with increasing concentrations of sucrose (IO%, 209;; and 30%) in 0. I M phosphate buffer for I-2 days until they sank. For electron-microscopy, the tissue blocks were left overnight in the buffer with 4”/6sucrose at 4°C. All sections (frozen for light-microscopy and vibratome for electron-microscopy) were cut at 60 pm in a plane perpendicular to the long axis of the hippocampus. The protocol for cytochrome oxidase histochemistry and ~ytochemistry was the same as reported previously.” All electron-microscope sections were cut near the surface of the blocks to ensure consistency in section depth and for the optimal localization of reaction products.

RESULTS The schematic diagram in Fig. 1 shows the location of the dentate gyrus and the various hippocampal fields (CAI, 2, 3 and 4), along with the basic laminar organization of each field. The trisynaptic pathway involving perforant path fibers, granule cells and their mossy fibers, CA3 pyramidal cells and their Schaffer collaterals, and CA1 pyramidal cells are drawn in to illustrate the pattern of synaptic relationship that the three principal cells have with each other and to show how the apical and/or basal dendrites of these cells are related

to the various

laminae

within

their

re-

the staining

in stratum moteculare of

CA1 was consistently intense in all species examined. but the relative levels of reactivity observed within the adjacent dentate outer molecular layer varied from moderate in squirref. monkey, gerbil and mouse to high in rat, cat and macaque. Likewise, the reactivity observed within stratum oriens varied from low and moderate in bat and rodent species to moderately high in rhesus and squirrel monkeys. One variation observed in this study appeared to be related to differences in the plane of sectioning. The transition in staining intensity between the outer and inner molecular layers varied from sharp (Figs 2,3, 5 and 8 INFRA) to gradual (Figs 4 and 8 SUPRA) not only within the same species, but also within the same animal or even the same section (Fig. 8). Lastly, although the same types of interneurons were observed in the different species, they were diilicult to observe in sections taken from brains that had been left in fixative for more than a few hours, or in sections that were not incubated long enough for clear differentiation. Despite these minor variations, consistent patterns of CO staining were discerned with respect to: (1) regions or fields, (2) laminae, (3) types of cells and (4) parts of the same cell (i.e. soma, dendrite, axon). Reyiod

distrihuriorl

qfccq’tochrorne oxictuse stuiniry

Although the most prominent light-microscopic observation in the hippocampus and dentate gyrus is a laminar distribution of cytochrome oxidase staining, there was a clear and consistent regional pattern of layer-specific reactivity within each hippocampal field (CA 1, 2, 3 and 4) and the dentate gyrus. The pattern of CO activity in the distal dendritic layers and in the somatic and proximal dendritic layers varied between different regions and so will be reported separately below. CA2 will be considered with CA3 since they exhibited similar levels of CO activity, although CA3 contains stratum lucidum and CA2 does not. The combined fields will be referred to as CA2--3 in this paper. In the somatic and proximal dendritic layers, the highest regional levels of CO activity were localized within CA2-3, while significantly lower levels were observed in CAI, CA4 and the dentate gyrus. In

Cytochrome oxidase activity in the hippocampus CA2-3, stratum pyramidale

and stratum oriens had relatively high levels of histochemical staining (Fig. 6, SP, SO). Above the pyramidal cell layer in CA3, a zone of low reactivity was observed which corresponded to stratum lucidum (Fig. 6, SL). In rodent species, a band of moderate reactivity was observed just above stratum lucidum within the lower half of stratum radiatum of CA3 (Fig. 6, SR). In CA1 and CA4 (Figs 7 and 8) the stratum pyramidale had low to moderate levels of reactivity with a few highly reactive presumed interneurons interspersed. Stratum oriens of CA1 also had low to moderate levels of activity and contrasted sharply with the higher levels observed in the same layer of CA2-3 (compare Fig.7 with Fig. 6). Stratum radiatum of CA1 consistently had low levels of histochemical staining (Fig. 7). In the dentate gyrus (Fig. 7, DG) the layer of granule cells was generally unreactive, while the layer of proximal dendrites (inner molecular layer,) exhibited low to moderate levels of CO activity as did the polymorphic cell layer. In the distal dendritic layers of CAl-3 and dentate gyrus, CO activity was notably more intense than in the somatic and proximal dentritic layers (Figs 6, 7 and 8). The most intense histochemical staining was localized within stratum moleculare of CA 1, followed by CA3 and the outer (213-314 depending on species) molecular layer of the dentate gyrus. Along the pial border of the dentate, the high reactivity of the outer molecular layer was intensified in the infra- but not suprapyramidal blade (Fig. 8 INFRA, SUPRA). In rodent species the intensity of staining in the infrapyramidal blade was often greater than that found in stratum moleculare of CA1 (Figs 2 and 5). Laminar distribution

Within each region of the hippocampus and dentate gyrus, CO activity was distributed in a layered pattern. The distal apical dendritic layers were the most intensely stained in all regions, while the somatic and proximal dendritic layers varied in the intensity of staining in accordance with the regional differences described above. Thus, in the hippocampus, the somatic (stratum pyramidale) and basal dendritic (stratum oriens) layers were generally more reactive than the proximal apical dendritic layer (Figs 6 and 7), while in the dentate gyrus the granule cell layer was less reactive than either the inner molecular or the polymorphic cell layers (Figs 7 and 8). Very low levels of CO activity were observed in stratum lucidum of CA3 and even lower activity was observed in the alveus. Principal cells and interneurons

In part, the regional and laminar distribution of cytochrome oxidase activity in the hippocampus and dentate gyrus represented the localization of activity in large populations of cells. One striking feature of this pattern of labeling in the cell body layers was that

2339

most of the cells in a given field appeared to have relatively the same level of activity which differed from that in the other fields. Hence, CA3 pyramidal cells were histochemically more reactive than CA1 and CA4 pyramidal cells, while dentate granule cells appeared to have little or no reactivity. Reactive proximal apical dendrites of CA3 pyramidal cells could be seen to traverse stratum lucidum in a course perpendicular to that of the unstained mossy fibers (Fig. 6, SL), while the proximal apical dendrites of CA1 pyramidal cells did not appear to be very reactive (Fig. 7, SR). Superimposed over the regional and laminar distribution of enzyme staining, many highly reactive cells were found scattered throughout most layers of the hippocampus and dentate gyrus (some are indicated by arrows, Figs 68). The reactive somata of many of these cells were observed in continuity with reactive varicose or smooth dendrites (Figs 15, 26, 28, 30 and 31) and resembled closely the shape and distribution of various interneurons described in Golgi preparations.35*48 Among these reactive interneurons, five types were distinguished in this study: (1) dentate pyramidal basket cells, (2) stratum pyramidale interneurons, (3) horizontal cells (fusiform cells are included here), (4) deep multipolar cells and (5) superficial multipolar cells. Reactive pyramidal basket cells were observed subjacent to the granule cell layer in the dentate gyrus (Figs 7, 14 and 26). Many of these cells had reactive apical dendrites that ascended through the overlying granule and inner molecular layer with diminished levels of reactivity. However, the distal varicose segments of these dendrites were highly reactive in the outer molecular layer. In the cat, these distal varicose dendritic segments were quite large and reacted very intensely for CO activity (Fig. 15, arrows). In stratum pyramidale of CA1 and 3, highly reactive somata of interneurons were frequently observed, sometimes in continuity with moderately reactive ascending varicose dendrites (Figs 30 and 31). Occasionally, a few of these varicose dendrites arborized in stratum moleculare with even greater reactivity. In the cat, these distal varicose dendrites were large and intensely reactive (Fig. 28, arrows). It is assumed that while many of these cells may be hippocampal pyramidal basket cells, some of them may correspond to the ‘pyramids with ascending axons’35 or to the ‘nonpyramidal’ interneurons (in stratum pyramidale) described by Schwartzkroin & Mathers6’ In this study these cells will be referred to as stratum pyramidale interneurons. In CA4, reactive multipolar cells were scattered throughout the expanded pyramidal cell layer (Fig. 8, arrows in CA4). A few of these cells had reactive proximal dendrites that extended in various directions. Since these cells were located within the principal cell layer of CA4, they may have been modified stratum pyramidale interneurons. Within the polymorphic cell layer of the dentate (Figs 7, 8, 14 and 26) and along the border between

2340

G. H. Kageyama

and M. T. T. Wong-Rile!

Fig. 1. Schematic diagram illustrating the major fields of the hippocampus. Small circles: granule cell bodies. Triangles: CAll4 pyramidal cell bodies. The three major cell types involved in the trisynaptic pathway of the hipocampus are drawn in to show the orientation of their dendrites and the projections of their axons. The distal dendrites of CA3 pyramidal cells also receive direct input hut is not shown here. Figs 2-5. Regional distribution of cytochrome oxidase activity. Cross sections of histochemically-reacted hippocampus and dentate gyrus. Black bars indicate boundaries between hippocampal fields. In all species, activity is highest above and below the hippocampal fissure and within the cellular and basal dendritic layers of CA3. Fig. 2. Rat hippocampus. The intensity of staining within the cellular layers is highest in CA3 followed by CA4, CA1 and the dentate gyrus. In the apical dendritic layers, the most intense reactivity is localized within CA1 and the lower blade of the dentate gyrus; however, the upper blade of the dentate and CA3 are also highly reactive.

( x 33)

Fig. 3. Macaque monkey hippocampus. The fields of the hippocampus in this plane are shifted in such a way that the fimbria can be seen projecting away from CA3 to the left. The relative size of CA3 is reduced, while the dentate is expanded compared with the rat. Despite these differences the overall pattern of histochemical staining is similar to that of the rat. (x 16) Fig. 4. Cat hippocampus. The reactive CA2 and CA3 pyramidal cells contrast sharply with the low activity in CA1 pyramidal cells and dentate granule cells. The apical dendritic layers have high levels of activity that do not significantly vary from field to field. (x 26) Abbreviations ALV, AP. CAll4, Den., DG, DMI, FIM, GC. GL, GRCL, HC. HF. I. IML INFRA, MF, MML, MT, OML PB, PC, PCl, PC3, PCL, PP. SA. SC, SL SM, SMI. SO, sP> SP, SPI, SR, SUPRA, TH, large ‘solid black arrows, large open arrows, small arrows, small arrows in Figs IS, 28, 30 and 31,

alveus apical dendrite cornu ammonis dendrite dentate gyrus deep multipolar

used in figures

subfields

l-4 of Lorente

de No3*

interneuron

fimbria granule cell glia limitans granule cell layer horizontal (or fusiform) cell hippocampal fissure interneuron inner molecular layer infrapyramidal blade of the dentate gyrus mossy fibers middle molecular layer mossy terminals outer molecular layer (includes MML in the text) pyramidal basket cell pyramidal cell pyramidal cell in CA1 pyramidal cell in CA3 polymorphic cell layer perforant path septal afferents Schaffer collaterals stratum lucidum stratum moleculare superficial multipolar interneurons stratum oriens dendritic spine stratum pyramidale stratum pyramidale interneuron stratum radiatum suprapyramidal blade of the dentate gyrus thalamohippocampal pathway reactive mitochondria non-reactive or weakly reactive mitochondria interneurons varicosities

in interneuron

dendrites

Cytochrome oxidase activity in the hippocampus Fig. 5. Woodrat hippocampus. The pattern of staining is similar to that in Figs 2-4. (x 26) The three white boxes indicate where subsequent photographs were taken at a higher magnification. Boxes from left to right represent Figs 68, respectively. Fig. 6. CA2 and CA3. Elevated levels of cytochrome activity are found in stratum moIeculare, stratum pyramidale and stratum oriens. The lower half of SR is moderately reactive. A band of low reactivity of SL is characteristic of CA3. Reactive superficial multipolar interneurons are found in SR and SL (arrows), deep multipolar interneurons in SO are unlabeled, and fusiform cells (arrow) are found along the SO/white matter border. (x 104) Fig. 7. CA1 and the dentate gyrus. The highest levels of cytochrome activity are localized within stratum moleculare of CA1 and the outer molecular layer of the dentate gyrus. The IML and PCL of the dentate as well as SO, SR and SP of CA1 have low to moderate levels of activity. In CAl, fusiforms cells are observed along the SO/ALV border, deep multipolar cells within SO, and SP interneurons in SP, but superficial multipolar interneurons were rarely observed in SR. In the dentate gyrus, reactive pyramidal basket cells were observed at the GRCL/PCL border, while horizontal and multipolar cells were found in PCL (not labeled). The black bar indicates where the hippocampal fissure separates CA1 from DG. (x 104) Fig. 8. CA4. Most of the cells within this field have low to moderate levels of CO activity, whereas a few scattered multipolar cells (arrows) are very reactive. These are presumed to be interneurons because of their shapes, sizes and distribution. Note the greater intensity of staining in the outer molecular layer of the infra- compared with the supra-pyramidal blade of the dentate. (x 104) Fig. 9. Dentate granule cells (GC). These cells have anly a narrow rim of cytoplasm with very few mitochondria, most of which are non-reactive. ( x 6050) Fig. 10. Outer molecular layer (OML) in the infrapyramidal blade of the dentate gyrus. High concentrations of reactive mitochondria were localised mainly within lucent dendritic profiles and within the astrocytes that form the glia limitans (GL). Axon terminals generally contained less reactive mitochondria. ( x 11,800) Fig. 11. Outer molecular layer (OML) in the suprapyramidal blade. High concentrations of reactive mitochondria are localized mainly within dendritic profiles of granule cells, while axon terminals generally contained less reactive mitochondria. ( x 11,800) Fig. 12. Middle molecular layer (MML). High concentration of reactive mitochondria are localized mainly within dendrites, while axon terminals contained nonreactive mitochondria. A reactive granule cell dendrite can be seen crossing the center of the field with a small dendritic spine (sp) budding off to the right. The spine forms an asymmetric synapse with a small axon terminal that contains a single non-reactive mitochondrion (upper open arrow). A larger spine (sp) containing a spine apparatus is seen below the first one. It is contacted by a non-reactive terminal (lower open arrow) at an asymmetrical synapse. ( x 11,800) Fig. 13. Inner molecular layer (IML). Mitochondria within this layer were less reactive and less concentrated than in the middle and outer molecular layers. Some reactive mitochondria are observed within proximal granule cell dendrites (solid arrow at lower left) and within small axodendritic synaptic terminals (solid arrow on the right). Axon terminals that formed synapses with dendritic spines, on the other hand, were usually non-reactive (lower two open arrows). (x 11,800) Fig. 14. Dentate gyrus. Four types of cells are shown: pyramidal basket, horizontal, superficial multipolar (I) and numerous granule cells within GRCL. The somata and proximal dendrites of the three types of interneurons are reactive while the somata of dentate granule cells have low activity. ( x 260) Fig. 15. Highly reactive varicose dendrites of presumed pyramidal basket cells and possibly other hilar interneurons within outer molecular layer. The reactivity of these dendrites diminishes proximally as they course through IML and GRCL. The continuity of these dendrites with reactive pyramidal basket cells was occasionally observed. ( x 221) Fig. 16. Dentate pyramidal basket cell (PB). A pyramidal basket cell contains numerous reactive mitochondria with an occasional non-reactive one. This cell is characterized by having an ovoid, extensively infolded nucleus, often with a prominent nucleolus and an intranuclear rod (inset a) or sheet. These cells also receive numerous axosomatic synaptic contacts. The one shown (inset b) contains reactive mitochondria. Granule cells, on the other hand, contain mainly non-reactive mitochondria. ( x 8600) Fig. 17. CAl-3 transition in the hippocampus. Within stratum pyramidale a clear transition in cytochrome oxidase reactivity was usually observed between CA1 and CA2-3. In CAl, pyramidal cells were non-reactive, however, reactive presumed interneurons (I) were scattered within this layer. In sharp contrast to CA1 pyramidal cells CA2 and CA3 pyramidal cells were highly reactive. One large reactive multipolar interneuron (I) is also shown within SO of CA3. SL appears as a clear band above the CA3 pyramidal cells. This is the layer of mossy fiber input from the dentate gyrus. Reactive proximal apical dendrites of CA3 pyramidal cells can be seen to extend across this layer. ( x 260)

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G. H. Kageyama and M. T. ‘T. Wong-Rile! Fig. 18. CAI pyramidal cell. A CAI pyramidal cell is shown on the lower left of the field. It contarns L\ population of non-reactive mitochondria. Likewise, the apical dendrites of these cells contain numerous microtubules, but low numbers of non-reactive mitochondria. In contrast. 3 small dumetcl dendrite, presumably from an interneuron. obliquely crosses the center of the field and contain< numerous reactive mitochondria and forms numerous synapses with axon terminals, One of thcsc axon terminals is shown to contain a single non-reactive mitochondrion (open arrow pointing to ~ynapsct sparse

(x 5800) Fig. 19. CA3 pyramidal cell. A CA3 pyramidal cell is shown to contain numerous reactive mitochondria and contrasts sharply with the CA1 pyramidal cell shown in Fig. 18. A few non-reactive mitochondria are also observed within this cell. (x 5800) Figs 20-23. Layers in CA1 and CA3 Fig. 20. Stratum moleculare (SM) of CAL High concentrations of intensely reactive mitochondria are localized mainly within dendrites. A few dendritic spines are shown-one is labeled (sp) and contains a spine apparatus. Mitochondria are not observed within these structures. Axon terminals usually contain a single non-reactive mitochondrion. (x 11.800) Fig. 21. Stratum radiatum (SR) of CAl. Very low concentrations of mitochondria are localized within the thick apical dendritic trunks (AP) of CA 1 pyramidal cells. The mitochondria shown within these structures are sometimes reactive (one is labeled with a solid arrow, far right), but usually they are not. Numerous synaptic contacts are formed in the neuropil with dendritic spines. One such spine (sp) is labeled and contains a spine apparatus. The mitrochondria within axon terminals in this layer were generally non-reactive. ( x 11,150). Fig. 22. Stratum Iucidum (SL) of CA3. The neuronal elements within this layer generally contain non-reactive mitochondria, with the exception of the large apical dendritic trunks of the CA3 pyramidal cells, which usually have several reactive mitochondria. ( x 11,800) Fig. 23. Stratum oriens (SO) of CA3. The most reactive mitochondria within this layer were concentrated mainly within dendritic profiles. Low to moderate levels of activity were observed in some of the axon terminals. ( x 1I, 150) Fig. 24. Stratum pyramidale interneuron (I) from CA3. Interneurons are characterized by the presence of an infolded nucleus, a prominent nucleolus, an intranuclear rod or sheet (narrow black arrow), and several axosomatic synaptic contracts (upper and lower solid arrows). These cells ccontained numerous reactive mitochondria. ( x 10,800) Fig. 25. Horizontal cell (HC) from the polymorphic cell layer of the dentate gyrus. Note the infolded nucleus and presence of numerous reactive mitochondria and dense bodies within the cytoplasm. These cells were also observed to contain intranuclear rods or sheets (not shown) and receive numerous axosomatic synaptic contacts. ( x 17.150) Fig. 26. Dentate interneurons-pyramidal basket (PB) and horizontal cell (HC) and numerous nonreactive granule cells in GRCL. (x 390) Fig. 27. Electron-micrograph of a highly reactive dendrite within the polymorphic cell layer. This dendrite presumably belongs to an interneuron. possibly a pyramidal basket or horizontal cell. This dendrite forms numerous synaptic contacts with axon terminals that contain non-reactive mitochondria. (x 11.140) Fig. 28. Reactive distal varicose apical dendrite of stratum pyramidale interneurons in CAI. Note the varicosities (arrows) of the reactive dendrite and how the reactivity within it is enhanced within SM. ( x 390) Fig. 29. Electron-micrograph of an intensely reactive varicose dendrite in stratum moleculare of CAl. corresponding to the type of dendrite shown in Fig. 28. These varicose dendrites receive numerous synaptic contacts all along the thick and thin portions of its length and contain a high concentration of intensely reactive mitochondria. The presynaptic axons generally have low reactive mitochondria. (x 11,750) Figs 3&31. Light-micrographs of reactive stratum pyramidale interneurons located within CAl. Several reactive varicose dendrites can be seen to ascend through stratum radiatum. Individual varicosities are indicated by arrows. Some reactivity extends into the basal dendrites as well. The intense staining of these ceils contrasts sharply with the relatively low reactive CA1 pyramidal cells within the same layer. The staining in some of these dendrites were observed to increase within stratum molecuiare (see Fig. 28). ( x 273) Fig. 32. Electron-micrograph of a reactive varicose dendrite within stratum radiatum (SR) corresponding to the middle dendritic segments of the stratum pyramidale interneurons shown in Figs 30 and 31. Numerous non-reactive axons terminals form synaptic contacts with both thick and thin segments. Reactive mitochondria in both the varicose dendrite and a principal cell dendrite are indicated by solid arrows. (x 7800)

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Plate 3 (Figs 9. IO). Dentate gyrus-granule cells. The electron-micrographs and most of the following light-micrographs are taken from cat hippocampus-reacted for cytochrome oxidase activity. The light-micrographs in Figs 14, 30 and 31 are taken from the woodrat, although the same types of reactive principal cells and interneurons are found in all species examined. In the electron-micrographs, reactive mitochondria are indicated by large solid black arrows and mitochondria with relatively low reactivity are indicated by open arrows. 2345

Plate 3 (Figs

11 13).Dentate gyrus-outer 33h

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Plate 5 (Figs 14-16). Dentate gym-interneurons, 2341

2348

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Plate 8 (Figs 24 27). Hippocampal

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2351

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+ + + high,

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Outer molecular layer, SUPRA Outer molecular layer, INFRA Inner molecular layer Granule cell layer Polymorphic cell layer

1. Summary

Relative level of cytochrome oxidase activity: + + + + very high, Abbreviations are the same as those used for the photographs.

Principal cells: Granule CA3 pyramidal CA 1 pyramidal Interneurons: Pyramidal basket Stratum pyramidale Horizontal (or fusiform) Deep multipolar (CAl-3) Superficial multipolar (CA3) Superficial multipolar (CA 1)

Cytological

Principal cell layers: Basal dendritic layers and polymorphic White matter:

Apical dendritic

Laminar

Distal apical dendritic layers Somatic/proximal dendritic layers

Regional

Table

gyrus

+

gyrus

+

+

+

nonreactive.

+++ +++ +++ +++ +++

+++

soma

SM SR SL SP so FIM

in the hippocampus

+++ ++++ ++ + ++

Dentate

+++

Dentate

activity

+ low, -

oxidase

proximal dendrites

+ ++ ++ ++ ++ ++ ’ +

++ ++

+++ ++ + +++ ++,+++

CA3

+++ ++,+++

CA3

ALV

+ + +,+

distal dendrities

+ +

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2354

Kageyama and M. T. T. Wong-Rile!

the alveus and stratum oriens (Fig. 6) reactive horizontal (or fus~form) cells were commonly observed. In these cells CO activity was detected throughout the somata into the proximal dendrites. Within the poiymorphic cell layer of the dentate and in stratum oriens of CAI-3, reactive deep multipolar cells were also observed (Figs 6 and 7). These cells varied considerably in size from small to large. One large, highly reactive deep multipolar cell (I) is shown at the bottom right of Fig. 17. Reactivity in these cells was also concentrated within the somata and proximal dendrites. Reactive superficial multipolar cells were commonly observed within stratum radiatum of CA3 but seldom within CA1 (Figs 6, 7, 14). They were also rarely observed in the outer molecular layer of the dentate. Since GAD-positive neurons had been reported in stratum radiatum of CA1,53 we examined Nissl counterstained preparations and observed that presumed interneurons were common there, but were not reactive for cytochrome oxidase. Reactive cells were seldom observed in stratum moleculare of CAI-3. ELECTRON MICROSCOPIC OBSERVATIONS The hip~campus of rat and cat were examined at the el~tron-microscopic level. Cytochrome oxidase activity was localized within the outer surface of the inner mitochondrial membrane, including the intracr&ate space, as reported before.” In the more reactive mitochondria, reaction products also filled the matrix. The highest concentrations of reactive mitochondria were observed in dendritic profiles within stratum moleculare (CAl-3) and the outer 2/3 molecular layer of the dentate gyrus. Moderately high concentrations of reactive mitochondria were also observed in the somata and proximal dendrites of CA3 pyramidal cells and several types of presumed interneurons. Some axon terminals that formed axosomatic synapses also contained reactive mitochondria. Relatively unreactive mitochondria were seen within the somata and proximal dendrites of granule cells and CA1 pyramidal cells. Many axospinous and axodendritic presynaptic terminals, myelinated and unmyelinated axons, and non-neuronal elements also contained fewer and less reactive mitochondria. Dentate

gyms

Granule and polymorphic cell layers. The small (7-lO,um diameter) somata of granule cells had very few mitochondria and seldom were any of them reactive (Fig. 9). Within or below the granuie layer, larger (15-25 Fm diameter) pyramidal basket cells were occasionally found (Fig. 16). These cells were identified by several ultrastructural features described by Ribak & Anderson5’ which included: an ovoid, extensively infolded nucleus often with a prominent nucleolus and intranuclear rod (Fig. 16a) or sheet and an organelle-rich cytoplasm. Most of the mitochondria within these cells were moderate to highly reactive. Large

axosomatic synaptic terminals assoctated with p!r;rmidal basket cells contained nunlerous reactive mm)chondria (Figs I6 and 16b). Synaptic terminals acsociated with the somata and proximal dendrites of granule cells were smaller but usually contained one or two reactive mitochondria. In the polymorphic cell layer, reactive mitochondria were observed within horizontal cells (Fig. 25) and dendrites of various interneurons (Fig. 27), but the mitochondria in axon terminals were notably less reactive. Molecular IaJer. The highest concentration of reactive mitochondria in the dentate was observed in the distal dendrites of granule and pyramidal basket cells located in the outer 2:3 molecular layer (OML. MML, Figs 10-12). Fewer and less reactive mitochondria were found in the proximal granule cell dendrites of the inner molecular layer (IML, Fig. 13). Throughout the molecular layer. dendritic spines were commonly identified by their continuity with a granule cell dendrite and/or by the presence of a spine apparatus (Fig. 12, sp). Reactive mitochondria were not observed to enter these tiny processes. These spines often formed asymmetric synapses with relatively non-reactive axon terminals (Fig. 12).

Strata py~a~l~d~~e. Within this cellular layer. numerous moderate to highly reactive mitochondria were observed in the somata of CA3 pyramidal cells (Fig. 19) and stratum pyramidale interneurons (CAI-3) (Fig. 24). In sharp contrast, the CA1 pyramidal cells generally had fewer mitochondria which were usually non-reactive (Fig. 18). In stratum pyramidale of CAl-3, an occasional axon terminal was seen to contact pyramidal cell bodies. They were small and sometimes contained one or two reactive mitochondria. Those axon terminals that contacted stratum pyramidale interneurons (Fig. 24) were usually larger with several highly reactive mito~hondria. Small diameter dendrites presumably belonging to stratum pyramidale interneurons had many reactive mitochondria and were observed to form numerous synaptic contacts with low reactive axon terminals (Fig. 18). Stratum rnoleculare (CAI-3). As in dentate gyrus, the highest numbers of reactive mitochondria were observed in the distal dendrites of stratum moleculare (CAI-3). Dendritic profiles were easily recognized by their pale cytoplasm, microtubular arrays and continuity with spinous processes. In this layer, both dendrites and dendritic spines were often observed in postsynaptic relation to axon terminals with low reactive mitochondria, while nearly ali dendritic protiles contained at least one or two highly reactive mitochondria (Fig. 20). Scattered varicose dendrites in this layer contained a large number of reactive mitochondria and formed numerous synapses also with low reactive axon terminals (Fig. 29). These dendritic profiles most likely represent the reactive distal varicose dendritic segments of stratum pyramidale inter-

Cytochrome oxidase activity in the hippocampus neurons observed in light-microscopic (Figs 28, 30, 31). Stratum

radiatum

(CAI-3)

and

preparations

stratum

lucidum

Within stratum radiatum the more proximal apical dendritic shafts of CA1 and CA3 pyramidal cells were easily recognized by their large diameter, vertical orientation, pale cytoplasm, parallel arrays of microtubules and sometimes continuity with thin spinous precesses (Fig. 21). In CA3, these large dendrites contained moderate amounts of reactive mitochondria compared with very low numbers found in the corresponding proximal dendritic segments of CA1 pyramidal cells. This was especially notable in stratum lucidum (Fig. 22) where CA3 dendrites receive synaptic input from mossy terminals. In contrast, the mitochondria within mossy fibers and mossy terminals in this layer were not usually very reactive. Likewise, within stratum radiatum of CAl-3, relatively few reactive mitochondria were observed within axons or presynaptic terminals. Reactive mitochondria were, however, observed in small diameter ascending varicose dendrites (Fig. 32) which presumably belong to stratum pyramidale interneurons. These dendrites formed numerous synaptic contacts with low reactive axon terminals. Stratum oriens. The basal dendrites of pyramidal cells (CAl-3) were considerably thinner than the apical dendrites of these cells and were observed to form occasional spinous contracts with low reactive axon terminals. In CA3, the basal pyramidal cell dendrites contained moderate to high concentrations of reactive mitochondria (Fig. 23) while lower concentrations were observed in CAl. Reactive dendrites of fusiform and multipolar interneurons were also occasionally observed within stratum oriens in association with numerous non-reactive axon terminals. Non-neuronal elements. The only non-neuronal elements that contained numerous reactive mitochondria were the astrocytes that formed the glia limitans (Fig. 10, GL) located in the very dense band along the outer edge of the infrapyramidal blade of the dentate gyrus (Fig 2, 4, 5, 8). Most of the perivascular astrocytes were non-reactive, although an occasional one may contain moderately reactive mitochondria. All other glial elements showed few and relatively nonreactive mitochondria.

(CA3).

DISCUSSION The present study indicates that different levels of cytochrome oxidase activity exist within the various regions, layers and cell types of the hippocampus and dentate gyrus (see Table 1 and Fig. 33 for summary of results). The consistent pattern of enzyme localization * In the 2-deoxyglucose studies cited above, autoradiographic grain density is noticeably more intense in the cellular and basal dendritic layers of CA3 than CAl. However, this feature was not mentioned by any of these authors.

2355

from species to species suggests that the metabolic organization of these structures is a very constant feature of the mammalian brain. Our results confirm earlier findings that different neuronal populations have relatively distinct patterns and levels of oxidative enzymatic activity.20,70 At the light-microscopic level, the regional and laminar distribution of highest CO activity is within the distal dendritic layers on either side of the hippocampal fissure (stratum moleculare of CAl-3 and outer molecular layer of the dentate gyrus) and within the somatic and basal dendritic layers of CA3. These are regions of major extrinsic afferent input to the hippocampus and dentate.8*g.13.24.47 These regions also have the most intense staining for succinate dehydrogenase (SDH)*‘s3* and the highest 2-deoxyglucose (2-DG) uptake 50*5g*indicating higher levels of metabolic activity there. This study demonstrates that anatomically and physiologically distinct neuron types within the same structure may exhibit a pattern and level of CO reactivity that is quite different from that of other neuron types. The apparent lack of interneuronal staining in an earlier study on the dentate gyrus5’ is unclear, but it may in part be related to the length of fixation time. In some of our own material, we have observed that more than a few hours of fixation time significantly reduces CO activity. We have also revealed that not only different parts of the same cell (e.g. soma, dendrite, axon) may have distinct levels of oxidative enzyme activity, but that CO reactivity may vary within different segments of the same dendrite. The differential patterns of CO reactivity will be discussed with respect to the known anatomy and physiology of the specific neuronal types. Principal

cells

Dentate granule cells. In the outer molecular layer of the dentate gyrus where numerous asymmetric axospinous contracts are found,43 the distal spiny dendrites of granule cells form the principal postsynaptic targets for the perforant pathway.g Stimulation of this pathway evokes large negative waves indicative of substantial excitatory postsynaptic potentials within the outer molecular layer.4,34*63 The localization of most of the intensely reactive mitochondria in the outer molecular layer correlates with the high level of histochemical staining there. The predominantly dendritic localization of reactive mitochondria in this layer suggests that the high degree of convergence of excitatory synapses from the active perforant pathway upon the distal segments of each granule cell dendrite may contribute to their rich oxidative metabolic activity. In contrast, the lower concentrations of progressively less reactive mitochondria within the proximal dendrites (inner molecular layer) and the granule cell somata (granule cell layer) suggests that the level of oxidative metabolic activity decreases proximal to the granule cell body. In these regions, i.e. the inner molecular and granule cell layers, there is a

predominance of inhibitory influences upon the granule cells, as evidenced by the maximal extracellular IPSP’ as well as low spontaneous activity in individual cells (l--2 spikes;s)h5* and the presence of numerous symmetrical synapses3’ many of which are GAD-immunoreactive.S~‘” The pattern of high levels of distal dendritic t’s low levels of proximal dendritic and somatic CO-reactivity concur with iit riuo physiological findings in the alert animal, that an elevated distal dendritic extracellular EPSP can coexist with depressed granule cell discharge in the dentate gyrus.68 A similar gating of granule cell discharge can be affected by septal stimulation,’ different behavioral states of the sleep-wakefulness cycle67.6* and the level of theta activity. I0 This is of considerable interest because the serotoninergic raphe nuclei and nor-adrenergic locus coeruleus which are involved in the regulation of sleep cycles42 and the cholinergic medial septal nuclei which is involved with the generation of theta activity46 all converge within the subgranular layer in the hilus of the dentate.33.4” where they presumably contact interneurons and thereby modulate the effectiveness of perforant path influence on granule cell discharge. CA3 pyramidal cells. Much like granule cells, CA3 pyramidal cells also have highly reactive distal dendritic segments specifically within the layer of perforant path input (stratum moleculare). In stratum radiaturn, where there is an absence of any major excitatory projection, CO activity is markedly reduced. The small region of moderately elevated reactivity within the lower half of stratum radiatum in CA3 coincides spatially with the distribution of the longitudinal (axial) association pathway.35 However. the physiological characteristics of this pathway are unknown to us. Within stratum lucidum, CA3 pyramidal ceils receive a massive convergent excitatory projection from the mossy fibers at the level of the proximal dendritic trunks. While this layer appears relatively non-reactive for CO activity at low magnification, it is actually traversed by vertically-oriented dendritic trunks of CA3 pyramidal cells, which contain numerous reactive mitochondria. These proximal dendritic trunks can be followed to the highly reactive somata of CA3 pyramidal cells. Even though large extracellular IPSPs are recorded in stratum pyramidale,3 the relatively intense CO staining of CA3 somata may be explained by the dominance of the proximally localized excitatory influence of the mossy (and presumably septofimbrial) fiber inputs. These inputs may also account, at least in part, for the moderately high rates of spontaneous firing (11-30 + spikes/s) in CA3 pyramidal cells.65 In stratum oriens, the basal dendrites of CA3 pyramidal cells are also quite reactive. It is known that these dendrites arborize within the afferent terminal field of the * It is presumed that the cells in the dentate gyrus with high (30-40 spikes/s) spontaneous activity were interneurons.

septohippocampal pathway’whore they form synapses2’ and presumably receive cholinergic”,” excitation ’ As a whole, the multimodal response properties of CA3 pyramidal cells appear IO he influenced by both perforant and septal afferent inputs. ” However. it is not clear what the rclativc intensities of synaptic inputs are for the various converging afferent systems upon these cells. Tentatively. we can state that CA3 pyramidal cells are most reactive where they receive their major afferent excitatory synaptic inputs from the perforant. mossy fiber and septohippocampal pathways, while they are least reactive where they do not (in stratum radiatum). CA! p_vran~iduI wlls. In stratum moleculare of C’Al, cytochrome oxidase staining was even more intense than within stratum moleculare of CA3 or the dentate outer molecular layer. This could be due to the convergence there of the (1) perforant. (2) crossed temporo-ammonic4x~h3 and (3) thalamohippocampa12’ pathways. Although the first two pathways are known to be excitatory sources of input to CA1 pyramidal cells.63 the physiological characteristics of the thalamohippocampal projection have not yet been investigated. Stratum radiatum contrasts sharply with the intense reactivity of stratum moleculare. The low CO activity within stratum radiatum is surprising since the CA1 pyramids presumably receive a major excitatory synaptic projection from the CA3 Schaffer collaterals in this layer. According to Vinogradova.” however. the rather unspecified and multimodal response properties of CA3 pyramids cannot be the source of the highly differentiated response properties of the CA1 pyramids. Instead, “CA3 works as a ‘valve’ or ‘trigger’, which only in some definite state (novelty, ‘mismatch’) allows the passage of a signal containing specific information through the CA1 neurons.“h5 Consistent with this hypothesis, the suppression of neurotransmission through the trisynaptic pathway during REM sleep and during passive and active awake behaviors (but not slow wave sleep)“,“’ may account for the relatively low CO activity within stratum radiatum. In stratum pyramidale and stratum oriens of C‘Al. the pyramids do not appear to receive any major proximally-localized excitatory synaptic input (unlike the CA3 pyramids). Thus. a predominance of inhibitory synaptic influences3.39,h’,62 in CA1 may account for the low spontaneous activity (3-10 spikes,‘s)6s and low CO reactivity in the somata and proximal dendrites of these cells. Although the level of CO reactivity within the distal apical dendrites of CAI pyramidal cells is intensified specifically within stratum moleculare, where they receive excitatory synaptic input from the perforant pathway, the physiological potency of this pathway has not been sufficiently studied in relationship to the alterations brought about by gating during specific behaviors. Hence, the distribution of high levels of cytochrome oxidase within this layer correlates also with the distribution of high capillary density.”

Cytochrome oxidase activity in the hippocampus intense 2-deoxyglucose uptake59 and elevated succinate dehydrogenase levels, *Obut it is not certain that this specific layer receives a greater amount of excitatory synaptic input. Interneurons The pattern of CO reactivity within the various types of interneurons differed in several ways from that in principal cells. First, interneurons were stained most intensely within their somata and proximal dendrites. Second, interneurons exhibited greater variance in the intensity of their staining than did principal cells. Third, some interneurons did not appear to have any reactivity at all. Unlike the principal neurons, the somata and proximal dendrites of the hippo~mpal interneurons receive numerous asymmetric (presumed excitatory) synapses, 21,52,64 Some of these cells (1) appear to receive massive proximally-localized excitatory synaptic input (20&30OH~),~’ (2) have high spontaneous firing rates (estimates of range vary from l@lOO and (3) can sustain high frespikes/s) ‘5+16*19*44*60*77 quency-evoked rates of discharge (400 Hz).‘~ These features of the hippocampal interneurons are consistent with their elevated levels of oxidative enzymatic activity mainly within their somata and proximal dendrites (this study) where they are presumed to receive a massive convergent excitatory synaptic input from recurrent collaterals of pyramidal cells3~30 and/or the cholinergic septofimbrial pathway.2Q*33~37*41~55 The increased intensity of CO staining within the distal varicose dendrites of pyramidal basket and stratum pyramidale interneurons occurs within the same zone of elevated reactivity in the distal apical dendritic segments of the principal cells, namely, within the axon terminal field of the perforant pathway. Although there is no strong anatomical or physiological evidence as yet for a direct perforant path-interneuron synapse, it is difficult to imagine any other major excitatory synaptic input that these dendrites could receive. The variation in the intensity of CO staining within interneurons correlates with the wide variation in their reported levels of spontaneous activity (10-100 spikes/s) (references cited above). However, it is not clear why some interneurons. especially those within stratum radiatum of CA1 are seldom reactive for cytochrome oxidase. One possible explanation is that unlike most other hippocampal interneurons, these do not appear to receive any major extrinsic excitatory synaptic input since they are not distributed within the axon terminal fields of the perforant or septohippocampal pathways. The GAD-immunor~ctivity of hippocampal interneurons (including the stratum radiatum interneurons in CA1)53 suggests that they are GABAergic, but the non-reactivity for cytochrome oxidase of stratum radiatum interneurons in CA1 implies that some CABAergic interneurons may not have high levels of oxidative metabolic activity. In contrast, the interneurons within the hippocampal

2351

system that do have intense CO staining are distributed mainly within the septohippocampal afferent terminal field and many of them appear to receive direct cholinergic septohippocampal input.2g733*3754’ This is consistent with the role of the medial septal nuclei as the pacemaker of theta rhythm in the hippocampus46 and the response property of many presumed interneurons called ‘theta units’49 to increase their moderately high discharge rates (l&40 spikes/s) to very high rates (30-100 spikes/s) during theta activity. ‘5,1*,’9,49 The high spontaneous discharge rates recorded from various interneurons imply that they are very active cells. The close correspondence between these cells and the CO reactive interneurons described in this study suggests that many of these cells have high levels of oxidative metabolic activity. Moreover, as we will discuss below, the axon terminals of many of these interneurons form symmetric axosomatic contacts and often contain CO reactive mitochondria. Axon term~nuls

Contrary to what one might expect, not all axonal terminals contain reactive mitochondria. In the neuropil of the mouse cortical barrel fields, both reactive and non-reactive axon terminals were found.72s76 In the cerebellum, presumed basket cell terminals around Purkinje cells contain highly reactive mitochondria (Wang-Riley” and our unpublished observations). These observations suggest that some axon terminals may have higher levels of oxidative enzymatic activity than others. Since many of the reactive axosomatic synaptic terminals in the cerebellum and hippo~mpus form symmetric (presumed inhibitory) contacts and are GAD-immunoreactive,5*s3 it is possible that synaptic terminals may have special energy requirements related to the type of neurotransmitter they use. On the other hand, since very high rates of spontaneous activity have been reported in various hippocampal interneurons, 1h,19730.60it indicates that levels of CO activity in axon terminals may be related to their frequency of activation. Myelinated and unmyeiinated axonal trunks

The relatively low levels of CO activity in axonal trunks is consistent with the low levels of SDH,ZO 2-deoxyglucose uptake50s59 and oxygen consumption5’ in white matter as compared to gray matter. The concentration of SDH in peripheral nerves only at the nodes of Ranvie?’ suggests that oxidative metabolic activity in axons is mainly concerned with the regulation of ion fluxes. Thus, in axons, low levels of oxidative metabolic energy may be attributed to the low actual amount of ion exchange across the axonal membranez5 and the high efficiency with which they conduct impulses. In terms of energy consumption, CreutzfeldtL2 estimated that spike activity accounts for less than 0.3-37, of the total energy metabolism of the brain.

2358 Correluriofl

C. H. Kageyama with presmed

and M. T. T. Wong-Rile?

neurotrmmitters

Although there is a positive correlation between the distribution of many CO reactive and GAD-immunoreactive neurons in the hippocampus, certain extrapyramidal nuclei and monkey striate cortex,23~““~‘4~‘0 some GAD-immunoreactive neurons such as cerebellar Purkinje cells and stratum radiatum CA1 interneuronss3 are not reactive for cytochrome oxidase (Wong-Riley;” this study). By the same token, nonGAD-positive neurons such as the presumed glutaminergic CA3 pyramidal cells” and choline acetyltransferase-immunoreactive (presumed cholinergic) red nucleus (magnocellular) and cranial motor nerve neurons29 are highly reactive for CO (Wong-Riley;” this study). It appears that intense CO reactivity, however, is present in GABAergic neurons that have high levels of spontaneous or evoked excitatory synaptic activity such as in the nucleus reticularis thalami (1S-60 spikes/s), substantia nigra, pars reticulata (2-60 spikes/s) and for most of the interneurons of the hippocampus (30-80 spikes/s)Z6.45,54.56 (our unpublished observations of CO activity). In the hippocampus and dentate, the main neurochemical relationship that can be made is that the highest levels of CO activity appear to be localized within two places: (1) dendrites (or dendritic segments) and/or somata that are in postsynaptic relationship to a high density of convergent or frequently-activated excitatory synapses mediated primarily by glutamate (perforant path and mossy fibers) and/or acetylcholine (septohippocampal pathway) ‘7,29.33 (this probably includes many presumed GABAergic interneurons, which appear to receive direct extrinsic afferent input from these excitatory pathways); and (2) axon terminals that form axosomatic contacts, many of which form symmetric contacts and are presumably GABAergic and inhibitory in function. This is not to rule out that certain axon terminals (such as those that form axosomatic contacts with interneurons) containing other transmitters and mediating, say. excitation are not CO-reactive. Excitatory

synaptic

actiuit?

One possible reason for the close correlation between CO activity and excitatory synaptic activity is that the excitatory postsynaptic potential (EPSP) involves the entry of Na+ which needs to be actively pumped out against a steep electrochemical gradient at the expense of significant amounts of metabolic energy. If the highest energy-consuming process in the brain is the active transport of ions (mainly Na+) for the maintenance of resting potentia132*36,57,58 and only a small proportion of this energy is related to spike activity,” then it would seem that most of the metabolic energy in neurons must be related to membrane repolarization (via the Na+/K+ pump) subsequent to EPSP depolarization. Thus, brain

metabolism as determined by O2 consumptio0’ or 2-deoxyglucose uptake6 is stimulated by membrane depolarization but not by hyperpolarization, and the magnitude of transient oxidations of cytochrome oxidase is increased by depolarizing stimulation.” Ocerall

cytochrome

osidasr

staining

irk murons

Although the maintenance of resting potential ma) account for a large portion of the CO level in neurons, a significant share may also be attributed to such processes as protein synthesis, fast axoplasmic and intradendritic transport, neurotransmitter recycling (i.e. synthesis. packaging, release, reuptake and/or degradation) and active transport of substances across the plasma membrane. The energy requirements of some of these processes may be influenced by the size of a neuron’s soma and its axonal and dendritic arborizations, as well as its spike frequency. The functional segregation of many of these activities to specific parts of a neuron (i.e. soma, dendrite, axon or axon terminal) may account for some of the intracellular differences in CO activity. Thus, the overall level of CO staining in neurons should reflect its total oxidative energy requirements demanded by all of its functional activities. Conclusions

Cytochrome oxidase activity can vary not only between different cell types, but also between soma and dendrites of the same cell, or different segments of the same dendrite. A single neuron can also have different patterns of reactivity in its apical aersus basal dendrites. High levels of CO reactivity within dendrites and somata are present where there is intense localized convergent and/or pathway-specific excitatory synaptic input. The degree of dendritic and somatic reactivity in the same cell may be quite independent of one another, due to the presence of potent proximally-localized excitatory or inhibitory control over somatic depolarization. It is the level of spontaneous or synaptically-evoked somatic depolarization that appears to be correlated with the intensity of CO staining in the somata of neurons. The positive correlation between CO activity and GAD-immunoreactivity in neurons presumably applies only to GABAergic neurons that have high levels of spontaneous or synaptically-evoked activity.

Acknowledgemenfs-We are grateful to C. Banka, P. Hunter, C. L. Lee, L. Moy and Dr H. J. Ralston III for supplying us with some of the animals. We are also indebted to D. Akers, E. Bever, G. Conchola and D. Crumrine for their skilled technical assistance and to B. Natareili for typing the manuscipt. Special thanks to I. Morrison and Dr G. R. Shepard from G. Kageyama for providing the inspiration for this work. This work was partially supported by National Institute of Health Grant NS-16429.

2359

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