340
Brain Research, 140 (1978) 340-343 © Elsevier/North-Holland Biomedical Press
The fate of synaptic receptors in the kainate-lesioned striatum
JOHN W. OLNEY and TASIJA de GUBAREFF Washington University School of Medicine Department of Psychiatry, Seattle, Wash. (U.S.A.)
(Accepted August 31st, 1977)
Kainic acid (KA) is a potent neuroexcitatory analog of glutamate (GLU) which exerts GLU-like dendritosomatotoxic activity against central neurons, but is a much more powerful neurotoxin than G L U itself 6. In 1975 we reported that KA, when microinjected directly into rat brain in low dosage (3.5 nmoles), destroys local nerve cells without damaging axons passing through or terminating in the injected area 7,8. We proposed that KA be employed as a lesioning tool for selectively deleting neuronal perikarya from specific brain areas without disturbing axons of passage a and predicted that neurons in many, if not most, brain regions would be found sensitive to the toxic action of KA and related excitotoxins 7. In the following year, Coyle and Schwarcz I and McGeer and McGeer 8 reported that a single injection of KA (5-10 nmoles) into rat striatum results in rapid loss of striatal neurons and specific changes in transmitter enzymes (~ glutamic decarboxylase, choline acetyitransferase, 1' tyrosine hydroxylase) similar to those known to occur in the striatum of patients with Huntington's Chorea. They suggested that the KAlesioned rat striatum might serve as a useful animal model for studying Huntington's Chorea. More recently, results of synaptic receptor binding studies and other biochemical measurements on the KA-lesioned rat striatum have begun to appear 1°, 11. Interpretation of such data is hampered by lack of correlative ultrastructural information pertaining to changes in synaptic connections induced by KA. Here we report the unexpected finding that, despite loss of all striatal neurons, many synaptic complexes comprised of both pre- and postsynaptic components remain intact in the KA-lesioned striatum three weeks after treatment. A total of 16 adult male Wistar rats (Harlan, Cumberland, Ind.) weighing 250 g were used in these experiments. Kainic acid (Sigma, St. Louis, Mo.) was introduced stereotaxically into the striatum through a 30 gauge cannula by the method of Swanson et al. 12. To achieve central placement of the cannula in the striatum, stereotaxic coordinates of Pelligrino and Cushman (AP 2.2, L 2.7, V 5.8) were employed 9. KA was prepared in low concentrations (10-20 mM) in sterile distilled H20 with enough N a O H and NaC1 added to yield an isotonic (154 mM) solution at neutral pH. Each animal, under diabutai anesthesia, was given a single injection of a fixed volume (0.5 #1) of the KA solution in either 10 or 20 m M concentration so as to
341 deliver either 5 or 10 nmoles of K A to the injected striatum. This volume was injected slowly over a 5-min period and the cannula was left in place for an additional 10 min before removal to prevent retrograde movement of the injected solution up the cannula tract. Animals were sacrificed 1, 2 or 3 weeks after injection by glutaraldehyde-paraformaldehyde perfusion fixation 4 under chloral hydrate anesthesia and three-quarter m m serial slabs collectively encompassing the entire striatum were additionally fixed in osmium tetroxide and processed for combined light and electron microscopic examination by methods previously described4, 7. In our experience 5, and this roughly correlates with the histological observations of Schwarcz and Coyle ii, a 5-nmole dose of K A rapidly destroys about. 70 % of the neurons in the striatum, and 10 nmoles destroys nearly all striatal neurons. K A does not exert toxic action upon axons of passage, however, so that even a 10-nmole K A injection which eliminates all neurons from the striatum causes degeneration of only those axons originating from the degenerating neurons. Axons of extrinsic origin remain healthy and in abundance in the striatum 1, 2 or 3 weeks following K A injection. Amorphous debris representing remnants of degenerating neurons can be detected in the neuropil 3 weeks after treatment, although the bulk of such material has been phagocytized and degraded beyond recognition by this time. Thus, except for
Figs. 1. a, b and c: the synaptic complexes depicted here are characteristic of those abundantly present in the striatum 3 weeks after a 10-nmole KA injection. They appear to be asymmetric synapses of the type which axon terminals ordinarily make with dendritic spines and shafts of striatal neurons. The presynaptie element appears healthy and contains numerous synaptic vesicles,mostlyof the clear variety but dense-core vesicles are also occasionally present. The synaptic cleft remains distinct and has postsynaptic dense material extending from it which does not appear fundamentaly different from the postsynaptic webb of an intact synapse (a = 96,000 x ; b and c -- 45,000 x).
342 necrotic material and glia or phagocytic elements, the KA-lesioned striatum at 3 weeks is found by electron microscopy to consist entirely of axons which vary in size and appearance from large-caliber myelinated axons to small-caliber preterminal and terminal axons. With one notable exception, there are no normal appearing nonaxonal neural elements present. The exception is that many axon terminals retain their postsynaptic membranes in relatively intact condition for up at least 3 weeks after treatment, even though such membranes no longer have any attachment to the dendritic spine, shaft or cell soma which originally housed them (Fig. 1). Examples of this phenomenon are so common in the KA-lesioned striatum at 3 weeks that several such synaptic profiles are routinely seen in every field scanned at 6000 ;
Brain Research, 140 (1978) 340-343 © Elsevier/North-Holland Biomedical Press
The fate of synaptic receptors in the kainate-lesioned striatum
JOHN W. OLNEY and TASIJA de GUBAREFF Washington University School of Medicine Department of Psychiatry, Seattle, Wash. (U.S.A.)
(Accepted August 31st, 1977)
Kainic acid (KA) is a potent neuroexcitatory analog of glutamate (GLU) which exerts GLU-like dendritosomatotoxic activity against central neurons, but is a much more powerful neurotoxin than G L U itself 6. In 1975 we reported that KA, when microinjected directly into rat brain in low dosage (3.5 nmoles), destroys local nerve cells without damaging axons passing through or terminating in the injected area 7,8. We proposed that KA be employed as a lesioning tool for selectively deleting neuronal perikarya from specific brain areas without disturbing axons of passage a and predicted that neurons in many, if not most, brain regions would be found sensitive to the toxic action of KA and related excitotoxins 7. In the following year, Coyle and Schwarcz I and McGeer and McGeer 8 reported that a single injection of KA (5-10 nmoles) into rat striatum results in rapid loss of striatal neurons and specific changes in transmitter enzymes (~ glutamic decarboxylase, choline acetyitransferase, 1' tyrosine hydroxylase) similar to those known to occur in the striatum of patients with Huntington's Chorea. They suggested that the KAlesioned rat striatum might serve as a useful animal model for studying Huntington's Chorea. More recently, results of synaptic receptor binding studies and other biochemical measurements on the KA-lesioned rat striatum have begun to appear 1°, 11. Interpretation of such data is hampered by lack of correlative ultrastructural information pertaining to changes in synaptic connections induced by KA. Here we report the unexpected finding that, despite loss of all striatal neurons, many synaptic complexes comprised of both pre- and postsynaptic components remain intact in the KA-lesioned striatum three weeks after treatment. A total of 16 adult male Wistar rats (Harlan, Cumberland, Ind.) weighing 250 g were used in these experiments. Kainic acid (Sigma, St. Louis, Mo.) was introduced stereotaxically into the striatum through a 30 gauge cannula by the method of Swanson et al. 12. To achieve central placement of the cannula in the striatum, stereotaxic coordinates of Pelligrino and Cushman (AP 2.2, L 2.7, V 5.8) were employed 9. KA was prepared in low concentrations (10-20 mM) in sterile distilled H20 with enough N a O H and NaC1 added to yield an isotonic (154 mM) solution at neutral pH. Each animal, under diabutai anesthesia, was given a single injection of a fixed volume (0.5 #1) of the KA solution in either 10 or 20 m M concentration so as to
341 deliver either 5 or 10 nmoles of K A to the injected striatum. This volume was injected slowly over a 5-min period and the cannula was left in place for an additional 10 min before removal to prevent retrograde movement of the injected solution up the cannula tract. Animals were sacrificed 1, 2 or 3 weeks after injection by glutaraldehyde-paraformaldehyde perfusion fixation 4 under chloral hydrate anesthesia and three-quarter m m serial slabs collectively encompassing the entire striatum were additionally fixed in osmium tetroxide and processed for combined light and electron microscopic examination by methods previously described4, 7. In our experience 5, and this roughly correlates with the histological observations of Schwarcz and Coyle ii, a 5-nmole dose of K A rapidly destroys about. 70 % of the neurons in the striatum, and 10 nmoles destroys nearly all striatal neurons. K A does not exert toxic action upon axons of passage, however, so that even a 10-nmole K A injection which eliminates all neurons from the striatum causes degeneration of only those axons originating from the degenerating neurons. Axons of extrinsic origin remain healthy and in abundance in the striatum 1, 2 or 3 weeks following K A injection. Amorphous debris representing remnants of degenerating neurons can be detected in the neuropil 3 weeks after treatment, although the bulk of such material has been phagocytized and degraded beyond recognition by this time. Thus, except for
Figs. 1. a, b and c: the synaptic complexes depicted here are characteristic of those abundantly present in the striatum 3 weeks after a 10-nmole KA injection. They appear to be asymmetric synapses of the type which axon terminals ordinarily make with dendritic spines and shafts of striatal neurons. The presynaptie element appears healthy and contains numerous synaptic vesicles,mostlyof the clear variety but dense-core vesicles are also occasionally present. The synaptic cleft remains distinct and has postsynaptic dense material extending from it which does not appear fundamentaly different from the postsynaptic webb of an intact synapse (a = 96,000 x ; b and c -- 45,000 x).
342 necrotic material and glia or phagocytic elements, the KA-lesioned striatum at 3 weeks is found by electron microscopy to consist entirely of axons which vary in size and appearance from large-caliber myelinated axons to small-caliber preterminal and terminal axons. With one notable exception, there are no normal appearing nonaxonal neural elements present. The exception is that many axon terminals retain their postsynaptic membranes in relatively intact condition for up at least 3 weeks after treatment, even though such membranes no longer have any attachment to the dendritic spine, shaft or cell soma which originally housed them (Fig. 1). Examples of this phenomenon are so common in the KA-lesioned striatum at 3 weeks that several such synaptic profiles are routinely seen in every field scanned at 6000 ;
