Abstract
Antonucci F, Alpár A, Kacza J, Caleo M, Verderio C, Giani A, Martens H, Chaudhry FA, Allegra M, Grosche J, Michalski D, Erck C, Hoffmann A, Harkany T, Matteoli M, Härtig W. J Neurosci 2012;32(6):1989–2001.
Inhibitory (GABAergic) interneurons entrain assemblies of excitatory principal neurons to orchestrate information processing in the hippocampus. Disrupting the dynamic recruitment as well as the temporally precise activity of interneurons in hippocampal circuitries can manifest in epileptiform seizures, and impact specific behavioral traits. Despite the importance of GABAergic interneurons during information encoding in the brain, experimental tools to selectively manipulate GABAergic neurotransmission are limited. Here, we report the selective elimination of GABAergic interneurons by a ribosome inactivation approach through delivery of saporin-conjugated anti-vesicular GABA transporter antibodies (SAVAs) in vitro as well as in the mouse and rat hippocampus in vivo. We demonstrate the selective loss of GABAergic—but not glutamatergic—synapses, reduced GABA release, and a shift in excitation/inhibition balance in mixed cultures of hippocampal neurons exposed to SAVAs. We also show the focal and indiscriminate loss of calbindin+, calretinin+, parvalbumin/system A transporter 1+, somatostatin+, vesicular glutamate transporter 3 (VGLUT3)/cholecystokinin/CB1 cannabinoid receptor+ and neuropeptide Y+ local-circuit interneurons upon SAVA microlesions to the CA1 subfield of the rodent hippocampus, with interneuron debris phagocytosed by infiltrating microglia. SAVA microlesions did not affect VGLUT1+ excitatory afferents. Yet SAVA-induced rearrangement of the hippocampal circuitry triggered network hyperexcitability associated with the progressive loss of CA1 pyramidal cells and the dispersion of dentate granule cells. Overall, our data identify SAVAs as an effective tool to eliminate GABAergic neurons from neuronal circuits underpinning high-order behaviors and cognition, and whose manipulation can recapitulate pathogenic cascades of epilepsy and other neuropsychiatric illnesses.
Commentary
Injury or dysfunction of GABAergic interneuron networks accompanies many types of epilepsy. One major scientific challenge in the field of neuroscience is to delineate the contributions of specific populations of GABAergic interneurons to cognition and neurological disorders. Local-circuit GABAergic inhibitory interneurons in the hippocampus and cerebral cortex regulate the timing and patterning of neural activity, and orchestrate the slow rhythmic oscillations within hippocampal–entorhinal cortex networks that mediate experience-dependent memory formation. Disruption of GABAergic interneurons’ roles in generating patterned activity may be partly responsible for learning disorders and cognitive impairments in autism and schizophrenia. Recent efforts to delineate these functional roles in the normal brain and in neurologic disease have employed genetic methods to eliminate different functional types of GABAergic interneurons during development (1), and genetic silencing or “reversible silencing” using optogenetics to control the firing of specific populations in GABAergic interneurons. Now, botulinum neurotoxin and saporin-based toxins have been added to the molecular arsenal for probing GABAergic interneuron functions and region-specific roles in epilepsy (2, 3).
Saporin, from the seeds of Saponaria officinalis, is a long-lasting eukaryotic ribosomal toxin that is resistant to proteolysis and denaturation. The prediction that saporin-mediated inactivation of ribosomes could be used to selectively target GABAergic interneurons in a region-specific manner was tested in a recent study by Antonucci and colleagues. By cross-linking saporin to antibodies against the C-terminus of vesicular gamma-amino-butyric acid transporter (VGAT), they selectively targeted inhibitory synapses. VGAT is found ubiquitously in GABAergic and glycinergic neurons, where it localizes to presynaptic vesicles in synaptic terminals and plays a role in accumulating GABA into synaptic vesicles (4). During vesicular fusion events when GABA is released into the synaptic cleft, the C-terminus of VGAT is transiently exposed at the surface of the presynaptic ending before the membrane undergoes endocytosis (5).
Hypothesizing that the transient extracellular presence of VGAT's C-terminus might provide a GABAergic neuron-specific target for saporin toxin, the scientists conjugated saporin to anti-VGAT-C antibodies, and designated the immunotoxin particles “SAVAs”. In a multi-lab effort led by Tibor Harkany in Sweden, Michela Matteoli in Italy, and Wolfgang Hartig in Germany, SAVA uptake in primary neuronal cultures of the hippocampus was shown to destroy inhibitory synapses and eliminate GABAergic interneurons, while leaving glutamatergic terminals and neurons intact. Moreover, electrophysiological recordings showed that SAVA treatment of primary hippocampal cultures reduced the frequency of postsynaptic inhibitory currents, while sparing the frequency and amplitude of excitatory postsynaptic currents. In a further test of their hypothesis in the intact brains of rodents, the scientists stereotaxically injected SAVAs or unconjugated anti-VGAT-C antibodies, as controls, into the CA1 region of the hippocampus before investigating the loss of GABAergic interneurons, using an array of molecular markers for the different interneuron subclasses. They found a striking loss of parvalbumin+ interneurons, eliminating the perisomatic inhibition to the principal neurons of CA1 from these cells. Moreover, virtually all subtypes of GABAergic interneurons were destroyed indiscriminately within this delimited region, including neuropeptide Y+, calbindin+, VGLUT3+, and somatostatin+ GABAergic interneurons. The lesions were sharply delineated and confined to CA1, leaving CA3 and the dentate gyrus intact. Accompanying these changes, microglia within CA1 became activated and phagocytic, a further confirmation of neuronal degeneration.
To evaluate whether SAVA-mediated elimination of GA-BAergic interneurons in CA1 circuits triggered epileptiform events, they performed EEG recordings 11 to 12 days after injecting the toxin into the hippocampus. Similar to their in vitro findings, removing inhibitory interneuron networks in CA1 generally increased neuronal activity in the hippocampus. High-amplitude EEG discharges were found in the hippocampus of immunotoxin-injected animals, but not the controls. Additionally, sporadic generalized seizures were observed in mice with SAVA-lesions when they were handled, suggesting that SAVA-mediated GABAergic interneuron loss in CA1 is sufficient to alter the synchronized activity and temporal control of hippocampal principal cells. While the EEG studies were only performed for short periods in the mice with SAVA lesions, these results are intriguing because they suggest that the mice may have developed a mild form of mesial temporal lobe epilepsy.
To further assess the similarities with temporal lobe epilepsy, they studied SAVA-injected mice after longer survivals of 10 weeks. At these time points, additional GABAergic interneuron losses were found in CA3 and the dentate gyrus, glutamatergic synapses thinned in strata oriens and radiatum, and granule cells dispersed in the dentate gyrus granule cell layer—similar to changes in hippocampal structures that occur in human patients and models of mesial temporal lobe epilepsy in rodents.
Despite the specificity of the initial damage to GABAergic interneurons in CA1, longer survivals resulted in delayed injury to pyramidal neurons, as well as GABAergic interneurons in other subfields of the hippocampus. For example, they also found that CA1 pyramidal neurons were depleted over time, resulting in an increase in high-amplitude and high-frequency discharges in the hippocampus, consistent with damage that occurs with recurrent seizures in mesial temporal lobe epilepsy.
This new strategy for creating selective lesions of GABAergic interneurons demonstrates that one immediate impact of removing CA1 interneurons is to increase high-amplitude epileptiform-like events. Although these events are milder than those observed in some other rodent models of mesial temporal lobe epilepsy, the subsequent loss of glutamatergic neurons and synapses, as well as granule cell dispersion, allows further temporal distinctions to be made between the development of spontaneous seizures and the various neuropathologic hallmarks of temporal lobe epilepsy. Moreover, the findings suggest that loss of inhibitory drive in hippocampal networks may trigger downstream excitotoxic damage. Further analyses will need to dissect the cellular and molecular events that lead to an escalation in damage to determine whether inflammation plays a critical role in pyramidal neuron degeneration and whether secondary loss of interneurons in the dentate gyrus is due to seizure-like events or another cause. Region-specific silencing of GABAergic interneurons with SAVAs may be a powerful new approach for investigating how GABAergic interneurons in different regions of the hippocampus regulate aspects of behavior. For example, how does the loss of inhibitory activity within CA1 affect theta/gamma rhythms and associated behaviors, including the formation of memories by the hippocampal–entorhinal cortex network? Additionally, as a potential model for mesial temporal lobe epilepsy, SAVA-treated rodents may be useful for short-term testing of potential anticonvulsant drugs, neuroprotective pharmaceutical compounds, or novel gene- and stem cell–based therapies for treating epilepsy.