Substance P receptor expression by inhibitory interneurons of the rat hippocampus: Enhanced detection using improved immunocytochemical methods for the preservation and colocalization of GABA and other neuronal markers

Abstract

Two unresolved issues regarding the identification and characterization of hippocampal interneurons were addressed in this study. One issue was the longstanding inability to detect gamma-aminobutyric acid (GABA) in the somata of several hippocampal interneuron subpopulations, which has prevented the unequivocal identification of all hippocampal interneurons as GABA neurons. The second issue was related to the identification of the hippocampal interneurons that constitutively express substance P (neurokinin-1) receptors (SPRs). The recent development of neurotoxins that specifically target SPR-expressing cells suggests that it may be possible to destroy hippocampal inhibitory interneurons selectively for experimental purposes. Although SPRs are apparently expressed in the hippocampus only by interneurons, colocalization studies have found that most interneurons of several subtypes and hippocampal subregions appear SPR-negative. Thus, the identities and locations of the inhibitory interneurons that are potential targets of an SPR-directed neurotoxin remain in doubt. Using newly developed methods designed to copreserve and colocalize GABA and polypeptide immunoreactivities with increased sensitivity, the authors report that virtually all hippocampal interneuron somata that are immunoreactive for parvalbumin (PV), calbindin, calretinin, somatostatin (SS), neuropeptide Y, cholecystokinin, and vasoactive intestinal peptide exhibited clearly detectable, somal, GABA-like immunoreactivity (LI). Hippocampal SPR-LI was detected only on the somata and dendrites of GABA-immunopositive interneurons. All glutamate receptor subunit 2-immunoreactive principal cells, including dentate granule cells, hilar mossy cells, and hippocampal pyramidal cells, were devoid of detectable SPR-LI, even after prolonged electrical stimulation of the perforant pathway that induced the expression of other neuronal proteins in principal cells. Thus, hippocampal interneurons of all subtypes and subregions were found to be SPR-immunoreactive, including the PV-positive interneurons of the dentate hilus and hippocampus, and the SS-positive cells of area CA1, both of which were previously reported to lack SPR-LI. Only minor proportions of hippocampal interneurons appeared clearly devoid of detectable SPR-LI. These results demonstrate for the first time that all identified interneuron subpopulations of the rat hippocampus are GABA-immunoreactive, and that many inhibitory interneurons of all subtypes in all subregions of the rat hippocampus express SPRs constitutively.

Glutamatergic input from specific sources influences the nucleus accumbens-ventral pallidum information flow

“…Perfusion and preparation of tissue sections After 2 weeks' survival time the animals were deeply anaesthetized using Equitesin and perfused through the heart first with saline (2-3 min) followed by a fixative containing 2% paraformaldehyde (Sigma) and 0.5% glutaraldehyde (TAAB) in 0.1 M sodium acetate buffer (pH: 6.5) for 2 min, followed by 2% paraformaldehyde/0.5% glutaraldehyde in 0.1 M sodium borate buffer (pH: 8.5) for 1 h (Sloviter et al 2001). Brains were removed from the skull.…”

Stereological analysis of GluR2-immunoreactive hilar neurons in the pilocarpine model of temporal lobe epilepsy: Correlation of cell loss with mossy fiber sprouting

“…Second, some GluR2+ hilar neurons may have been inhibitory interneurons rather than mossy cells. Previous studies suggest that this error should have been small, especially since the rabbit antibody developed by Petralia et al (1997) was used (Sloviter et al, 2001;Bender et al, 2003). Third, it is possible that only the top of the cell penetrated the counting frame in some instances, causing the cell to be misclassified as a granule cell-like GluR2-immunoreactive hilar neuron.…”

“…Because the excitatory mossy cells are intermixed with inhibitory hilar interneurons, a specific marker was required. The best available at present is GluR2 immunoreactivity (Leranth et al, 1996;Sloviter et al, 2001Sloviter et al, ,2003. Practically all telencephalic glutamate neurons express the GluR2 subunit of the AMPA receptor.…”

“…Some inhibitory neurons also express GluR2, but usually to a much lesser degree than glutamate neurons. Sloviter et al (2001) reported that <1% of the GluR2-immunoreactive (GluR2+) neurons in the dentate hilus were also immunoreactive when subjected to a sensitive "mirror" method for visualizing GABA. Moreover, when the number of GluR2/3-immunoreactive hilar neurons and hilar neurons that expressed GAD67 mRNA (presumptive inhibitory interneurons) were determined in the same rats, the total accounted for 98.4% of all hilar neurons .…”

Neurokinin1 Antagonists Potentiate Antidepressant Properties of Serotonin Reuptake Inhibitors, Yet Blunt Their Anxiogenic Actions: A Neurochemical, Electrophysiological, and Behavioral Characterization

“…Possible Involvement of Gabaergic, Glutatamergic, and Adrenergic Mechanisms NK 1 receptors have been identified on GABAergic neurones surrounding 5-HT cell bodies in the DRN, and studies in the septum and cortex suggest that NK 1 receptor antagonists may indirectly excite serotonergic neurones via a reduction of inhibitory GABAergic tone (Sloviter et al, 2001;Ma and Bleasdale, 2002;Stacey et al, 2002;Szeidemann et al, 1995;Ebner et al, 2008). This would enhance their responsiveness to SSRIs, by analogy to GABA B antagonists (Millan, 2006;Cremers et al, 2007).…”