Focal inhibitory interneuron loss and principal cell hyperexcitability in the rat hippocampus after microinjection of a neurotoxic conjugate of saporin and a peptidase-resistant analog of Substance P

Episodes of prolonged seizures or head trauma produce chronic hippocampal n etwork hyperexcitability hypothesized to result primarily from inhibitory i nterneuron loss or dysfunction. The possibly causal role of inhibitory neur on failure in the development of epileptiform pathophysiology remains uncle ar because global neurologic injuries produce such a multitude of effects. The recent finding that Substance P receptors (SPRs) are expressed exclusiv ely in the rat hippocampus by inhibitory interneurons provided the rational e for attempting to ablate interneurons selectively by using neurotoxic con jugates of SPR ligands and the ribosome inactivating protein saporin that s pecifically target Substance P receptor-expressing cells. Whereas intrahipp ocampal microinjection of a conjugate of native SP and saporin produced sig nificant nonspecific damage at concentrations needed to produce even limite d selective loss of SPR-positive cells, a conjugate of saporin and the more potent and peptidase-resistant SP analog [Sar(9), Met(O-2)(11)] Substance P (SSP-saporin) caused negligible nonspecific damage at the injection site, and a virtually complete loss of SPR-like immunoreactivity (LI) up to 1 mm from the injection site. Within the SPR depletion zone, immunoreactivities for most GABA-, parvalbumin-, somatostatin-, and cholecystokinin-immunorea ctive cells and fibers were eliminated. The few interneurons detectable wit hin the affected zone were devoid of SPR-LI. The apparent loss of interneur ons was selective in that calbindin- and glutamate receptor subunit 2 (GLuR 2) -positive principal cells survived within the affected zone, as did myel inated fibers and the extrinsic calretinin- and tyrosine hydroxylase-immuno reactive terminals of subcortical afferents. An apparent lack of reactive s ynaptic reorganization in response to interneuron loss was indicated by zin c transporter-3 (ZnT3)- and beta -synuclein-LI, as well as by Timm staining , all of which revealed relatively normal patterns of excitatory terminal d istribution. Control injections produced minor damage at the injection site , but no apparent specific loss of SPR-LI. One to 12 weeks after injection of SSP-saporin, extracellular electrophysiological field responses recorded in the CA1 pyramidal and dentate granule cell layers in response to affere nt stimulation were blindly evaluated simultaneously in two sites 1-2 mm ap art along the longitudinal hippocampal axis. SSP-saporin-treated rats exhib ited relatively normal responses in some sites, whereas disinhibition and h yperexcitability indistinguishable from the pathophysiology produced by exp erimental status epilepticus were simultaneously recorded at adjacent sites . Anatomic analysis of the recording sites in each animal revealed that epi leptiform pathophysiology was consistently observed only within areas of SP R ablation, whereas relatively normal evoked responses were recorded from i mmediately adjacent and relatively unaffected regions. These data establish the efficacy of [Sar(9), Met(O-2)(11)] Substance P-saporin for producing a selective and spatially extensive ablation of hippocampal inhibitory inter neurons in vivo and a highly focal disinhibition that was restricted to the site of interneuron loss. These results also demonstrate that the "epilept ic" pathophysiology produced by experimental status epilepticus or head tra uma can be replicated by focal interneuron loss per se, without involving p rincipal cell loss and other interpretive confounds inherent in the use of global neurologic injury models. J. Comp. Neurol. 436:127-152, 2001. (C) 20 01 Wiley-Liss, Inc.