Injection construct for the generation of allatostatin (AL) neuropeptide receptor (AlstR)/tetO mice. The cDNA encoding the AlstR transgene was excised with BamHI and XbaI and ligated into the multiple cloning site of the pTRE-tight vector (Clontech). The resulting plasmid was cut with XhoI to yield a fragment containing the pTRE element driving the entire coding sequence of the allatostatin receptor followed by the SV40 polyadenylation site. The 2 pairs (inner and outer) of genotyping primers used were OF: 5′-CACTGGAAACGGTAGTATC-3′; OR: 5′-CGTGACTCTGCGGAAGG-3′; IF: 5′-GGATCACAATGCCAACGAC-3′; IR: 5′-CAGATCTCCTCCTCCGTG-3′.

A and B: AL significantly decreased intrinsic membrane excitability in neurons from +/+ (B) but not −/− (A) mice as evidenced by the increase in the number of action potentials elicited across a range of depolarizing current injections. Black represents recordings acquired in control artificial cerebrospinal fluid (ACSF); red represents recordings acquired following bath application of AL. C and D: consistent with G-protein-coupled inward rectifier K⁺ (GIRK) activation, bath application of AL significantly hyperpolarized the membrane (C) and reduced the resting input resistance (D) of CA1 pyramidal neurons from +/+ mice only.

A: multiunit spiking responses to white noise bursts across a range of stimulus intensities (0–80 dB SPL). Each dot is the spike count (mean ± SE, n = 20 repetitions) in a 100-ms window following sound onset. Same recording site and animal as Fig. 1. Black line represents saline superfusion 35 min prior to allatostatin; red line represents 12 min after start of AL superfusion; blue line represents 19 min after start of 2nd saline superfusion. Note that spiking responses were silenced even at the highest stimulus intensities used (80 dB SPL). B: multiunit spiking responses across the same stimulus intensities in 3 +/+ mice (error bars indicate SE with n = 3 mice). Responses during AL (red line) were recorded at maximal silencing (12, 5, and 2 min after start of AL application). C: multiunit spiking responses across the same stimulus intensities, in 3 −/+ or −/− mice (n = 3 mice).

Robust penetrance of allatostatin receptor-associated mRNA was observed in principal neurons of the hippocampus in contrast to an almost complete lack of receptor-associated mRNA in hippocampal interneurons. A: 1X bright-field photomicrograph of a coronal section from the hemisphere opposite that used for in vitro characterization of silencing resulting from bath-application of AL. B: a composite of 10× dark-field photomicrographs illustrating fluorescent Nissl labeling in the hippocampus. The composite corresponds to the rectangular outline illustrated in A. C: a composite of 10× bright-field photomicrographs illustrating mRNA labeling of principal neurons in cornu ammonis 1 and 3 (CA1 and CA3, respectively), granule cells of the dentate gyrus (GrDG), and fasciola cinereum (FC). D: a magnified view of the rectangular region identified in B and C illustrating a semi-transparent view of fluorescent Nissl labeling overlain on the corresponding view of mRNA expression. Numbers in D indicate the number of soma co-expressing AlstR-associated mRNA and fluorescence (numerator) compared with the total count of fluorescing cells (denominator). hf: hippocampal fissue; LMol: lacunosum moleculare; Mol: molecular layer; Or: stratum oriens; Rad: stratum radiatum.

A: multiunit spiking responses to white noise bursts (70 dB SPL, 25 ms) before, during, and after 10 μM AL application. Recordings are from middle layers of primary auditory cortex. Each dot is the spike count (mean ± SE, n = 20 repetitions) in a 100-ms window following sound onset. The horizontal line for each dot indicates the duration of each stimulus protocol. Responses are normalized to the first measurement in the series. Note that spiking responses are markedly diminished within ∼2 min and completely silenced shortly thereafter. Note also that evoked responses began to recover even before the end of AL application and showed a rebound effect after ∼10 min of saline application. B–G, top row: extracellular voltage waveforms at each of the time points indicated in A. White noise bursts (interleaved intensities) were delivered at a rate 2/s. Bottom row: poststimulus time histograms at the same time points (70 dB bursts). , sound presentation. H and I: photomicrographs of AlstR mRNA in situ hybridization of a coronal slice from the brain of this mouse. Primary auditory cortex is located in the boxed region, which is expanded in I.

AL receptor-associated mRNA expression was observed in principal neurons throughout the forebrain. The most striking exception to this expression pattern was the complete lack of expression in neurons of the thalamus. A: AlstR mRNA-expressing neurons were observed in visual (V1) and somatosensory (S1BF) cortices, the entorhinal cortex (MEnt), and components of the amygdalar complex (CxA, BMP, AHi, PMCo). B: AlstR mRNA-expressing neurons were observed, in addition to the aforementioned regions, in the piriform cortex (Pir), olfactory tubercle (Tu), dorsal endopiriform cortex (DEn), subiculum (Sub), and retrospenial cortex (RSC). C: AlstR mRNA-expressing neurons were observed, in addition to the aforementioned regions, in the granule cell layer of the olfactory bulb (GrO), accumbens (Acb), nucleus of the lateral olfactory tract (LOT), and components of the amygdalar complex (AAV, AHiPM, MeA, MePV). D: AlstR mRNA-expressing neurons were observed, in addition to the aforementioned regions, in components of the olfactory system (AOL, LO) and the claustrum (Cl). mRNA expression was completely absent in thalamic nuclei (e.g., DLG, MGD, Po, VPM) as well as in the cerebellum (e.g., Crus1). E: AlstR mRNA-expressing neurons were observed, in addition to the aforementioned regions, in the anterior cingulate cortex (CG1, CG2), indusium griseum (IG), and the lateral septum (LS). F: AlstR mRNA-expressing neurons were observed, in addition to the aforementioned regions, in the retrosplenial cortex (RSA, RSG), components of the ventral hypothalamus (VH), and components of the amygdalar complex (BLA, BMP, BLV, PMCo, PLCo). aca: anterior commissure, anterior; acp: anterior commissure, posterior; cc: corpus callosum; fmj: forceps major; opt: optic tract; rf: rhinal fissue. 1× photomicrographs illustrate whole coronal and sagittal sections. Outlines (gray) indicate locations of 5× photomicrograph composites.