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(Stroke. 1996;27:1411-1416.)
© 1996 American Heart Association, Inc.

Articles

GABAergic and Asymmetrical Synapses on Somata of GABAergic Neurons in CA1 and CA3 Regions of Rat Hippocampus

A Quantitative Electron Microscopic Analysis

Zhi B. Yao, MD, PhD; Xiaoda Li Zao C. Xu, MD, PhD

the Department of Neurology, University of Tennessee at Memphis.

Correspondence to Zao C. Xu, MD, PhD, Department of Neurology, University of Tennessee at Memphis, Memphis, TN 38163. E-mail zxu@utmem1.utmem.edu.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
Background and Purpose CA1 pyramidal neurons in hippocampus die while CA3 neurons survive after transient ischemia. The imbalance of excitation and inhibition may contribute to this selective vulnerability. The purpose of this study was to examine the morphological basis of the above hypothesis.

Methods Male Wistar rats were perfused with 4% paraformaldehyde and 0.2% glutaraldehyde in 0.15 mol/L phosphate buffer. Coronal sections (50 µm) cut on a microtome were processed for {gamma}-aminobutyric acid (GABA) immunocytochemistry. Sections for electron microscopy were postfixed in 0.5% osmium tetroxide and embedded in high-viscosity epoxy resin. Ultrathin sections were cut and observed with an electron microscope.

Results GABA-positive neurons in the stratum pyramidale received more GABAergic synapses than asymmetrical synapses. The percentage of somatic membrane of GABA-positive neurons covered by asymmetrical synapses in the CA1 region (3.17±1.13%) was higher than that in the CA3 region (2.15±0.18%, P<.05). The ratio of asymmetrical to GABAergic synapses per 10 µm somatic membrane in the CA1 region (0.71±0.22) was higher than that in the CA3 region (0.53±0.14, P<.05). The ratio of the percentage of somatic membrane covered by asymmetrical/GABAergic synapses in the CA1 region (0.33±0.14) was also significantly higher than that in the CA3 region (0.20±0.07, P<.05).

Conclusions The GABAergic neurons in the CA1 region receive stronger excitatory inputs than those in the CA3 region, which provides a morphological basis for differences in excitability that may contribute to selective vulnerability after transient ischemia.


Key Words: cerebral ischemia • excitotoxicity • GABA • selective vulnerability • rats


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
In the central nervous system, GABA is an important inhibitory neurotransmitter. GABAergic interneurons form inhibitory synaptic connections with pyramidal cells in the hippocampus.1 2 These interneurons receive excitatory inputs from axon collaterals of pyramidal neurons and commissural afferents.3 4 5 GABAergic interneurons also receive inhibitory inputs from other inhibitory neurons, forming complex GABA-GABA connections.1 6 7 The balance between excitation and inhibition of GABAergic neurons determines their excitability, which in turn influences the activity of pyramidal neurons. In some neurological disorders, such as cerebral ischemia, the balance between excitation and inhibition of pyramidal neurons may have direct impact on their outcomes.

It has been well established that delayed cell death occurs in CA1 but not in CA3 pyramidal neurons after transient forebrain ischemia.8 9 The mechanisms underlying such selective vulnerability to ischemia are not well understood. Excitotoxic effects triggered by excessive glutamate release during ischemia have been hypothesized to be the major cause of postischemic neuronal injury.10 On the other hand, extracellular levels of GABA also significantly increase during ischemia.11 The increase of extracellular GABA during ischemia may inhibit neuronal activities and attenuate neuronal damage.12 13 GABA-mimetic drugs have been demonstrated to protect CA1 hippocampal neurons from ischemic insults.14 15 16 17

It has been shown that GABAergic neurons in the hippocampus are resistant to transient ischemia.18 19 20 However, neurophysiological studies indicate that the IPSPs, which are likely mediated by GABAergic interneurons, are more sensitive to ischemia than the EPSPs. The IPSPs evoked from pyramidal neurons are suppressed earlier than EPSPs during ischemia/anoxia21 22 23 and recover later than EPSPs after reperfusion.24 Congar et al25 recently reported that the excitatory inputs on interneurons in hippocampus are more rapidly depressed by anoxia than pyramidal cells. The authors suggest that the depression of IPSPs in hippocampus during anoxia is due to the functional disconnection of inhibitory interneurons from excitatory inputs. The loss of IPSPs in the hippocampus has been shown to be associated with the hyperexcitability of CA1 pyramidal cells.26 Therefore, the balance of excitation and inhibition in GABAergic interneurons may greatly influence the excitability of pyramidal neurons in the hippocampus. Differences in GABAergic local circuitry between CA1 and CA3 regions may contribute to the selective vulnerability of hippocampal neurons after transient ischemia.

One recent study has shown that CA3 pyramidal neurons receive more GABAergic innervation than CA1 neurons.27 This suggests that CA3 neurons may have stronger inhibition than CA1 neurons to counterbalance the excitotoxic effects during and after transient ischemia. To further reveal the morphological basis of selective vulnerability of pyramidal neurons to ischemic insult, we examined the excitatory and inhibitory inputs on GABAergic neurons in CA1 and CA3 regions of rat hippocampus. The GABAergic (inhibitory) and asymmetrical (excitatory) synapses on the somata of GABAergic interneurons in CA1 and CA3 regions were compared by means of immunocytochemical techniques with quantitative electron microscopy.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
Immunocytochemistry and Electron Microscopy
Experimental procedures in this study were performed within National Institutes of Health guidelines (Guide for the Care and Use of Laboratory Animals, NIH publication 93-23, revised 1985). Male Wistar rats (weight, 250 to 350 g) were anesthetized with ketamine (0.1 mL/100 g) and perfused transcardially with 0.01 mol/L sodium phosphate–buffered saline, followed by 400 mL of 4% paraformaldehyde plus 0.2% glutaraldehyde dissolved in 0.15 mol/L sodium phosphate buffer solution. The brain was removed, and a block containing hippocampus (5x5 mm) was cut and postfixed in 4% paraformaldehyde at 4°C for 2 hours. The block was cryoprotected in 30% sucrose for 20 hours, frozen in liquid nitrogen for 20 seconds, and then put in 0.01 mol/L KPBS at room temperature. Coronal sections (50 µm) were cut on a microtome (Vibratome, PELCO 101, Ted Pella Inc), collected in chilled KPBS, and rinsed three times. The sections were put in 1% sodium borohydride in KPBS at room temperature for 30 minutes and rinsed three times in KPBS before incubation in 10% normal horse serum in KPBS at room temperature for 2 hours. Then the sections were incubated for 48 hours with mouse monoclonal anti-GABA antibody (Chemicon Inc) at 1:200 dilution in 5% normal horse serum in KPBS at 4°C. After the sections were rinsed in KPBS, they were incubated with 1:200 biotinylated horse anti-mouse antiserum (Vector Labs) and 10% normal horse serum in KPBS for 10 hours at 4°C, followed by 1:100 avidin-biotin peroxidase complex (Vector Labs) for 1 hour. The sections were rinsed in 0.05 mol/L Tris buffer (pH 7.6) and incubated in 0.05% diaminobenzidine tetrahydrochloride in 0.05 mol/L Tris buffer containing 0.01% hydrogen peroxide for 8 to 12 minutes. All incubations were performed with continuous gentle agitation. After immunocytochemical procedures, the sections were postfixed in 0.5% osmium tetroxide in 0.15 mol/L phosphate-buffered saline for 1 hour and infiltrated with a high-viscosity epoxy resin embedding medium (Araldite/Embed-812, EMS) and mounted on slides coated with liquid releasing agent (EMS). The areas containing GABA-positive neurons in the pyramidal layer of CA1 and CA3 regions were remounted on plastic blocks and sectioned with an ultramicrotome. The ultrathin sections were collected with slotted grids or mesh grids coated with polyvinyl formal resin (Formvar, Monsanto Co), stained with lead citrate, and observed under an electron microscope (JEOL 1200).

Data Collection and Analysis
Six rats were used for the present study. Samples were taken from the CA1 and CA3 regions of dorsal hippocampal sections corresponding to a plane approximately 3.8 mm caudal to bregma.28 Only the somata with similar shapes located in or adjacent to the stratum pyramidale were selected for analysis. A total of 22 GABA-positive neurons were analyzed (12 in CA1 and 10 in CA3). The counts of GABAergic and asymmetrical synapses on each soma were performed on a montage microphotograph at a final magnification of x15 000. We measured the perimeter of the cell bodies and the length of synaptic contacts on the somata by tracing the membranes of the somata and the profile of postsynaptic specialization with NIH Image (version 1.57). The border between the somatic and dendritic membrane was defined as the point where the spherical shape of the somatic membrane was broken by the origin of a dendrite. If the somatodendritic transition was not clear, the boundary between soma and proximal dendrite was defined as the point where the width of the proximal dendrite corresponded to the minor diameter of the nucleus. The density of GABAergic and asymmetrical synapses on the soma of GABA-positive neurons was indicated as the number of synapses per 10 µm of somatic membrane. We measured the length of GABAergic and asymmetrical synaptic contacts on the somata by tracing the direct apposition of presynaptic terminals with membrane specialization. The total length of each type of synaptic contact on each soma was divided by the perimeter of cell body to estimate the percentage of GABAergic and asymmetrical synaptic coverage on the somata of GABA-positive neurons.

The quantitative difference of all the measurements between CA1 and CA3 regions was examined with the unpaired t test (Statview, version 4.0). Differences between asymmetrical and GABAergic synapses on each soma of GABA-positive neurons were examined with the paired t test.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
Under electron microscopy, GABA-positive neurons were identified by the presence of electron-dense reaction products that were evenly distributed in the cytoplasm and the nucleus. As shown in Fig 1ADown, many organelles and rough endoplasmic reticulum were found in the perikaryal cytoplasm of GABA-positive neurons. The complex invagination of the nuclear membrane was found in every GABA-positive neuron. The somata of GABA-positive neurons were encompassed by numerous immunoreactive terminals (Fig 1ADown). Most of these terminals formed synaptic contacts on the somata. The electron-dense reaction products were distributed throughout the terminals and often obscured the fine structures. However, pleomorphic presynaptic vesicles could be identified in lightly labeled terminals. The postsynaptic specialization of GABA-immunoreactive terminals was characterized by small discrete patches, which indicated that they were symmetrical synapses (Gray's II, Fig 1BDown). In addition to theseGABAergic synapses, some nonimmunoreactive terminals were found to form synapses with somata of GABA-positive neurons. The dense postsynaptic specialization and the small, round presynaptic vesicles associated with these terminals indicated that these unlabeled synapses were asymmetrical synapses (Gray's I, Fig 1CDown).



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  		Figure 1. GABAergic and asymmetrical synapses on somata of GABA-positive neurons in hippocampus. A, Example of GABA-positive neurons in hippocampus. The electron-dense immunocytochemical reaction products were evenly distributed in the cytoplasm. Many GABA-positive terminals (solid arrows) and some unlabeled terminals (open arrows) formed synaptic contacts on the soma of this neuron. B, Two GABA-positive terminals forming synaptic contacts on the soma (S) of a GABA-positive neuron. The discrete patches of postsynaptic specialization indicate that this is a symmetrical synapse (solid arrows). Despite the electron-dense immunoreactive products, the densely packed presynaptic vesicles can be identified. C, An unlabeled terminal forming a synapse on the soma (S) of a GABA-positive neuron. The dense postsynaptic specialization indicates that this is an asymmetrical synapse (open arrow).

More GABAergic synapses were found on each GABA-positive soma than asymmetrical synapses, at 7.75±1.91 versus 5.25±1.42 (mean±SD, n=12, P<.05) in CA1 and 10.10±3.60 versus 5.10±2.08 (n=10, P<.05) in CA3 regions (Fig 2Down). Because the somata size of GABA-positive neurons in CA3 was slightly larger than that in CA1 (Table 1Down), we compared the axosomatic synapses on GABA-positive neurons between CA1 and CA3 using the number of synapses per unit somatic membrane (10 µm). As indicated in Table 1Down, no significant difference was found in the number of GABAergic and asymmetrical synapses per 10 µm somatic membrane of GABA-positive neurons between CA1 and CA3 regions. However, the percentage of somatic membrane covered by asymmetrical synapses was higher in CA1 (3.17±1.13%) than in CA3 (2.15±0.18%, P<.05). The ratio of asymmetrical to GABAergic synapses per 10 µm somatic membrane in CA1 (0.71±0.22) was significantly higher than that in CA3 (0.53±0.14, P<.05, Table 2Down). The ratio of the percentage of somatic membrane covered by these two types of synapses in CA1 and CA3 was also significantly different (0.33±0.14 for CA1, 0.20±0.07 for CA3; P<.05, Table 2Down).



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  		Figure 2. Number of GABAergic and asymmetrical synapses on each soma of GABA-positive neurons in CA1 and CA3 regions. Values are mean±SD (n=12 in CA1, n=10 in CA3). The number of asymmetrical synapses on each soma is significantly lower than GABAergic synapses (P<.05, paired t test) in both regions.


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  		Table 1. GABAergic and Asymmetrical Synapses on Somata of GABA-Positive Neurons


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  		Table 2. Ratio of Asymmetrical to GABAergic Synapses on Somata of GABA-Positive Neurons


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 down arrowIntroduction 
 down arrowReferences 
 
The principal finding of the present study is that the GABA-positive neurons in the stratum pyramidale of the CA1 region receive more putative excitatory inputs than those in CA3, which provides a morphological basis for differences in excitability between these two regions.

Many types of GABAergic neurons, such as basket cells, chandelier cells, and bistratified cells, have been identified in the hippocampus.2 29 30 31 Most GABAergic interneurons in the stratum pyramidale are basket cells, in which a majority of their axon terminals contact somata and proximal dendrites of pyramidal cells.29 31 The GABA-positive neurons in the present study are similar in location and morphology to basket cells. Hippocampal GABAergic interneurons mediate two types of inhibition onto pyramidal cells. First, interneurons receive recurrent collaterals of pyramidal cells and in turn inhibit pyramidal cells in a form of feedback inhibition.5 Second, these interneurons are innervated by the commissural afferents from the contralateral hippocampus to form feed-forward inhibition.4 6 The asymmetrical synapses on GABA-positive neurons observed in the present study are probably formed by these two types of excitatory inputs. The observation that GABA-positive neurons receive GABAergic axosomatic synapses confirms the results of previous light microscopic studies. It has been shown that GABA immunoreactive terminals cover somata and processes of GABA-positive neurons in hippocampus.1 7 32 The GABAergic terminals on GABA-positive interneurons may come from other GABAergic interneurons within the region or from long association-commissural projections in CA1 and CA2.33 The results of the present study indicate that more GABAergic synapses are formed on the somata of GABA-positive neurons than asymmetrical synapses. Similar results have also been found in GABA-positive neurons of the dentate gyrus in gerbils.34 The soma is the most important part of a neuron to integrate inputs and influence outputs. The balance of GABAergic and asymmetrical synapses in the soma of a GABAergic neuron has powerful modulatory effects on the spike activity of the neuron, which in turn affects the target cells.

The functional significance of GABA-GABA interaction is disinhibition.35 36 The activity of GABAergic neurons in hippocampus depends on the balance of their excitatory and inhibitory inputs. The ratio of excitatory to inhibitory synapses on the somata reflects the excitability of GABAergic neurons and can be regarded as the "excitatory index" of inhibitory interneurons. The present study indicates that the ratio of asymmetrical to GABAergic synapses on somata of GABA-positive neurons in the CA1 region is higher than that in the CA3 region, ie, GABAergic neurons in the CA1 region possess a higher "excitatory index" than those in the CA3 region. Higher excitation of GABAergic neurons means stronger inhibition of pyramidal cells. This may partially explain the fact that CA3 pyramidal neurons are more seizure-prone than CA1 neurons.37 38 However, the balance between excitation and inhibition in GABAergic neurons is altered during ischemia/anoxia. It has been shown that the excitatory postsynaptic currents are more rapidly depressed by anoxia in interneurons than in simultaneously recorded pyramidal neurons.25 Such a functional disconnection of inhibitory interneurons from excitatory inputs may contribute to the higher sensitivity of IPSPs to ischemia/anoxia than EPSPs evoked in pyramidal neurons.21 22 39 40 Because the GABAergic neurons in the CA1 region receive stronger excitatory inputs than their counterparts in the CA3 region, the functional disconnection of these excitatory inputs during ischemia may cause the GABAergic neurons in CA1 to be less excitable than those in the CA3 region. Therefore, the inhibition of pyramidal neurons may be reduced more in CA1 than in CA3 after ischemia. It has been reported that the spontaneous firing rate in CA1 increases after ischemia, whereas the neuronal activity in CA3 decreases.41 Using brain slice preparation, Kawasaki et al42 also reported that hypoxia-induced seizures were usually initiated in and restricted to the CA1 region, whereas only 2.5% of these slices generated seizures in the CA3 region. The results of the present study provide a morphological basis for the above neurophysiological observations, which may be associated with the selective vulnerability of neuronal damage after transient forebrain ischemia.


*    Selected Abbreviations and Acronyms
 
EPSPs = excitatory postsynaptic potentials
GABA = {gamma}-aminobutyric acid
IPSPs = inhibitory postsynaptic potentials
KPBS = potassium phosphate–buffered saline


*    Acknowledgments
 
This study was supported by grants from the National Institutes of Health (NS33103) and the American Heart Association (94008130). We thank E. Howard for his assistance.

Received January 10, 1996; revision received March 8, 1996; accepted April 1, 1996.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
down arrowIntroduction 
 down arrowReferences 
 

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Editorial Comment

A Quantitative Electron Microscopic Analysis

John T. Povlishock, PhD, Guest Editor

Department of Anatomy, Medical College of Virginia, Virginia Commonwealth University, Richmond, Va


*    Introduction 
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 up arrowReferences
 *Introduction 
down arrowReferences 
 
In the accompanying article Yao and colleagues examine, through quantitative immunocytochemical and electron microscopic approaches, the proportion of GABAergic and purported excitatory input to the GABAergic interneurons supplying the CA1 and CA3 subsectors of the hippocampus. Through the assessment of the inhibitory and excitatory balance to the somatic domains of these GABAergic interneurons, the authors note that the interneurons supplying pyramidal neurons within the CA1 subsector receive a stronger excitatory input than those supplying the CA3 pyramidal neurons. On the basis of this finding, it is posited that this differential input forms the morphological basis for the differential excitability that contributes to the selective vulnerability seen in CA1 neurons after transient cerebral ischemia.

From an anatomic perspective, the present studies appear reasonably interpreted, although there is some concern regarding the assertion that all asymmetrical synapses must be excitatory. In this context, the report would have benefited from a more detailed characterization of the asymmetrical synapses, the use of appropriate immunocytochemical controls, and the incorporation of varied double-labeling strategies to identify multiple neurotransmitter populations. The present study's focus on the somatic domain rather than the dendritic tree is appropriate in that, as the authors acknowledge, the neuronal soma plays a major role in integrating synaptic input and influencing output. Clearly, this communication increases our knowledge of the synaptic input to the GABAergic interneurons modulating the CA1 and CA3 pyramidal neurons. This allows us to appreciate that differences in synaptic input may allow for differential modulatory control of the CA1 and CA3 pyramidal neurons, which may be related to their failure in the case of transient ischemia and seizure, respectively.

Notwithstanding these rather positive statements, there is legitimate concern that the authors may have overstated their findings and their relevance for understanding the pathobiology of transient ischemia. For example, although there may be differences between the ratio of asymmetrical to GABAergic synapses on the somata of the CA1/CA3 interneurons, it is unreasonable to assume that this difference alone contributes to all the differential vulnerabilities seen downstream in the target CA1 and CA3 pyramidal neurons. For example, the present communication does not consider the fact that the number of GABAergic neurons projecting to and/or the number of synaptic contacts made between these GABAergic interneurons and the pyramidal cells may also be factors in this pathobiology. For example, the authors have recently reported that the CA3 pyramidal neurons receive more GABAergic innervation than the CA1 neurons,1R suggesting that this difference may also play a role in their differential vulnerability.

Furthermore, while it is clear that the balance of excitatory and inhibitory input is a factor in the differential vulnerability of the CA1 and CA3 pyramidal neurons, there are other factors that should have been considered. As has been well explicated by multiple authors and recently reviewed by Siesjo et al,2R multiple postsynaptic factors may also be involved in the differential response of the pyramidal neurons to transient ischemia. This differential response may be linked to sustained derangements of calcium homeostasis that may in turn result in sustained alterations of plasma membrane function and transcriptional and/or translational changes. Clearly, the article would have benefited from the inclusion of such considerations to provide the readership with a more comprehensive appreciation of the complex pathobiology involved in the selective neuronal vulnerability seen within the CA1/CA3 pyramidal neurons.


*    Selected Abbreviations and Acronyms
 
EPSPs = excitatory postsynaptic potentials
GABA = {gamma}-aminobutyric acid
IPSPs = inhibitory postsynaptic potentials
KPBS = potassium phosphate–buffered saline

Values are mean±SD.

*Unpaired t test.


*    References 
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 up arrowReferences
 up arrowIntroduction 
 *References 
 

  1. Yao ZB, Li ZC, Xu ZC. Different distribution of GABAergic components between CA1 and CA3 region in rat hippocampus. Soc Neurosci Abstr. 1995;393:17. Abstract.
  2. Siesjo BK, Katsura K-I, Zhao QI, Folbergrova J, Pahlmark K, Siesjo P, Smith M-L. Mechanisms of secondary brain damage in global and focal ischemia: a speculative synthesis. J Neurotrauma.. 1995;12:943-956.[Medline] [Order article via Infotrieve]




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