Donald R. Gehlert

Delayed c-fos proto-oncogene expression in the rat hippocampus induced by transient global cerebral ischemia: an in situ hybridization study

The relative levels of c-fos mRNA in individual neurons of the hippocampal formation of rats is dramatically increased following 20 min of cerebral ischemia induced by 4-vessel occlusion. After 24 h of recirculation, a number of scattered neurons in the dentate hilus became hybridization positive. This effect appeared to peak between 24 and 48 h. A few neurons in the pyramidal cell layer of CA1 expressed c-fos as early as 24 h, but the most intense labeling in this region was seen at 72 h of recirculation. These results correlate well with the known distribution of delayed ischemic necrosis in the brain.

Muscarinic antagonist binding site heterogeneity as evidenced by autoradiography after direct labeling with [3H]-QNB and [3H]-pirenzepine

Saturable, high affinity binding of tritiated pirenzepine [( 3H]-PZ) was obtained in slide mounted tissue sections prior to performing autoradiographic localization of these binding sites. The binding in tissue sections of rostral rat forebrain gave a KD of 18nM and a Bmax of 51 fmoles/mg tissue. These binding characteristics are similar to those previously obtained in homogenate membrane preparations and indicate the binding is taking place in a similar manner. The distribution of the binding sites labeled with [3H]-PZ represented a subpopulation of those which could be labeled with tritiated quinuclidinyl benzilate [( 3H]-QNB). Thus, [3H]-PZ and [3H]-QNB both label regions of the cerebral cortex, hippocampus, striatum and dorsal horn of the spinal cord, while sites in the cerebellum, nucleus tractus solitarius, facial nucleus and ventral horn of the spinal cord are labeled with [3H]-QNB and not by [3H]-PZ. These observations indicate separate regions of the brain where antagonists bind to subtypes of muscarinic receptors.

Angiotensin II receptor localization in the canine CNS.

Specific binding of [125I]angiotensin II [(125I]Ang II) to sections of dog brain was determined by in vitro receptor autoradiography. Highly discrete, dark images representing specific binding of [125I]Ang II were observed in areas corresponding to the nucleus of the solitary tract, dorsal motor nucleus of the vagus, area postrema, ventrolateral medulla, pineal, subfornical organ, nucleus medianus, septum, organum vasculosum of the lamina terminalis and the anterior pituitary. The specific binding was frequently present either as a narrow band or tiny spot within a small portion of the nuclei to which the binding corresponded. The location of these Ang II recognition sites in regions associated with regulation of autonomic and neuroendocrine function provides further evidence for a role of this peptide within the central nervous system.

Quantitative light microscopic autoradiography of [3H]hemicholinium-3 binding sites in the rat central nervous system: a novel biochemical marker for mapping the distribution of cholinergic nerve terminals

The distribution of specific [3H]hemicholinium-3 ([3H]HC-3) binding sites throughout the rat forebrain was studied by means of quantitative light microscopic autoradiography. Tissue sections were labeled with 2.5 nM[3H]HC-3, apposed to tritium-sensitive film for 2 months and analyzed by computer-assisted densitometry. Regions of intense [3H]HC-3 labeling include the caudate-putamen, nucleus accumbens, olfactory tubercle, amygdala, habenula and the granule cell layer of the dentate gyrus. Little or no specific binding was detected in the corpus callosum, a white matter region. This distribution of specific [3H]HC-3 binding sites is compatible with a selective labeling of central cholinergic nerve terminals.

Quantitative autoradiography of [3H]forskolin binding sites in the rat brain.

The binding sites for a radiolabeled form of the potent activator of adenylate cyclase, forskolin, have been localized in the rat brain, pituitary and spinal cord. Using the quantitative technique of in vitro autoradiography, a high density of [3H]forskolin binding was detected in brain structures such as the caudate-putamen, nucleus accumbens, olfactory tubercle, globus pallidus, substantia nigra and the hilus of the area dentata. A comparison of the distribution of [3H] forskolin binding sites with those reported for several neurotransmitter receptor types indicated that forskolin identified adenylate cyclase was probably not linked to any single type of neurotransmitter receptor. These results also presented several new brain areas in which to investigate the neuronal role of adenylate cyclase.

γ-aminobutyric acidB receptors in the rat brain: Quantitative autoradiographic localization using [3H](−)-baclofen

The light microscopic localization of gamma-aminobutyric acidB (GABAB) receptors in the rat brain has been accomplished using in vitro autoradiography with [3H]baclofen as a ligand. Initial biochemical studies indicated this compound specifically bound to slide-mounted tissue sections with high affinity in a saturable and readily reversible manner. Autoradiograms generated by labeled tissue sections demonstrated high grain densities over structures such as the interpeduncular nucleus, molecular layer of the cerebellum, superior colliculus, several thalamic nuclei and the superficial laminae of the cortex. The majority of the GABAB sites were found to exist in regions of the brain previously reported to have significant densities of GABA sites present. A few areas were noted, however, where the distribution of GABAA and GABAB sites did not overlap.

Preclinical Evaluation of Melanin-Concentrating Hormone Receptor 1 Antagonism for the Treatment of Obesity and Depression

The mammalian neuropeptide, melanin-concentrating hormone, interacts with two G protein-coupled receptors, melanin-concentrating hormone receptor (MCHR) 1 and MCHR2; however, only MCHR1 is expressed in rats and mice. In the present study, we evaluated MCHR1 antagonism in preclinical models believed to be predictive of antiobesity and antidepressant activity. Central activity of the selective MCHR1 antagonist, GW803430 [6-(4-chloro-phenyl)-3-[3-methoxy-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-3H-thieno[3,2-d]pyrimidin-4-one], was evaluated using ex vivo binding with autoradiography. Effective doses of GW803430 (1 and 3 mg/kg p.o.) were correlated with antiobesity activity in a 14-day study of diet-induced obese rats. GW803430 was evaluated subsequently for antidepressant-like effects in mice and rats. Acute and subchronic administration reduced immobility time in the mouse forced-swim test at doses of 3 (acute) and 3 and 10 (chronic) mg/kg p.o., an effect that was absent in MCHR1(-/-) mice. Combined subeffective doses of GW803430 (0.3 and 1 mg/kg p.o.) and imipramine (5 mg/kg) produced a robust antidepressant-like response. The compound was also active in the tail suspension test at a dose of 10 mg/kg p.o. GW803430 (30 mg/kg p.o.) significantly reduced submissive behaviors at weeks 2 and 3, a model of submissive behavior that may predict antidepressant onset. GW803430 decreased marble burying in mice at doses of 3, 10, and 30 mg/kg p.o., an assay that detects anxiolytic-like effects. Thus, GW803430 produces robust antiobesity and antidepressant-like effects in rats and mice at doses that compete for central MCHR1 in vivo. As such, MCHR1 should be considered as a promising target for future drug discovery efforts.

Quantitative autoradiography of angiotensin II receptors in the SHR brain.

Several lines of evidence indicate brain angiotensin II is associated with the elevation of blood pressure seen in the spontaneously hypertensive rat (SHR). These include an increased pressor response to intracerebroventricularly administered angiotensin II and a reduction of blood pressure in response to centrally administered angiotensin II receptor antagonists. Using quantitative receptor autoradiography, we have detected greater angiotensin II receptor binding in a number of discrete brain nuclei of the 6-week-old SHR when compared to age-matched Wistar-Kyoto controls. Tissue sections from various brain regions were labeled with [125I]-angiotensin II according to a previously described method. Autoradiograms were generated by apposing the labeled tissue sections to LKB Ultrofilm along with brain paste standards which contained known amounts of [125I]. Quantitation of the binding, utilizing computer-assisted microdensitometry, indicated greater [125I]-angiotensin II binding in several brain areas implicated in cardiovascular control including the subfornical organ, nucleus of the solitary tract, dorsal motor nucleus of the vagus, locus coeruleus, supraoptic nucleus and the organum vasculosum of the lamina terminalis. Scatchard analysis of the binding in the nucleus of the solitary tract indicated an increased receptor number (Bmax) was responsible for the change while binding in two forebrain structures, the subfornical organ and supraoptic nucleus, showed alterations in receptor number and affinity (Kd). Several other brain regions, unrelated to cardiovascular control, exhibited no change in [125I]-angiotensin II binding. Since the increased receptor binding was present primarily in brain regions related to cardiovascular control, we conclude that an increased angiotensin II receptor affinity and density is indicated as a factor in the etiology of the high blood pressure seen in the SHR.

Vasopressin metabolite, AVP4–9, binding sites in brain: Distribution distinct from that of parent peptide

Binding sites for the vasopressin metabolite peptide, (AVP4-9), were detected in the rat brain. These binding sites were present in the hilus of the hippocampal formation, superior and inferior colliculus, pontine reticular nuclei, brainstem nuclei, lateral mammillary nucleus, choroid plexus and subfornical organ. The distribution of AVP4-9 binding sites was distinct from that of the parent peptide (1-3). This distinction was apparent in both the regional and intra-regional distribution.

Autoradiographic localization of “peripheral-type” benzodiazepine binding sites in the rat brain, heart and kidney

SummaryThe localization of peripheral-type benzodiazepine binding sites has been accomplished using quantitative receptor autoradiography after labeling slidemounted tissue sections with [3H]-RO 5-4864. Specific [3H]-RO 5-4864 binding sites were observed in the choroid plexus and ependyma cells within the brain. Lower levels of specific binding were seen in areas corresponding to the glomerular layer of the olfactory bulb. Stereotaxic administration of the excitotoxin, ibotenic acid, into the piriform cortex produced a dramatic increase in binding to the peripheral-type site in this brain region. Binding in the kidney was associated with the ascending limb of the loop of Henle and the distal convoluted tubule, while in the heart a more diffuse binding was found throughout the ventricle wall. The localization of peripheral-type benzodiazepine binding sites to areas involved in ion transport gives additional support to previously suggested physiological roles for these sites.