Semi-automated atlas-based analysis of brain histological sections

Quantifying the location and/or number of features in a histological section of the brain currently requires one to first, manually register a corresponding section from a tissue atlas onto the experimental section and second, count the features. No automated method exists for the first process (registering), and most automated methods for the second process (feature counting) operate reliably only in a high signal-to-noise regime. To reduce experimenter bias and inconsistencies and increase the speed of these analyses, we developed Atlas Fitter, a semi-automated, open-source MatLab-based software package that assists in rapidly registering atlas panels onto histological sections. We also developed CellCounter, a novel fully automated cell counting algorithm that is designed to operate on images with non-uniform background intensities and low signal-to-noise ratios.     

Characterization and autoradiographic localization of TRH receptors in sections of rat brain

Receptors for thyrotropin-releasing hormone (TRH) in rat brain have been localized autoradiographically by exposing tritium-sensitive film to sections labeled with [3H]3-Me-His2-TRH. Greatest grain density was found over certain nuclei of the amygdala, with considerable density over several other forebrain areas. Properties of TRH receptor binding in frozen sections closely resembled those of receptors in fresh membrane fragments.     

Localization and characterization of 35S-t-butylbicyclophosphorothionate binding in rat brain: an autoradiographic study

35S-t-butylbicyclophosphorothionate (TBPS) binding to slide-mounted rat brain sections was characterized for subsequent autoradiographic analysis. Cortical brain mash slices, preincubated with EDTA to remove endogenous GABA, were used for biochemical characterization. Steady state for 35S-TBPS binding was reached by 3 hr of incubation at 22 degrees C. The association rate constant (K1) and dissociation rate constant (K2) were 0.377 min-1 microM-1 and 0.011 min-1, respectively. Dissociation was monophasic and slow (t1/2 = 80 min). The kinetically derived KD was 29.4 nM. Scatchard analysis indicated a single population of binding sites with a KD of 21.0 +/- 2.2 nM and a Bmax of 1.59 +/- 0.13 pmol/mg protein. Both picrotoxin and muscimol inhibited 35S-TBPS binding completely with IC50s of 251 +/- 13 nM and 203 +/- 41 nM and nHs of 0.98 and 1.4, respectively. The distribution of 35S-TBPS binding sites in the rat brain resembles that of other ligands that bind to GABAA receptor complex with some regionally specific differences. Regions with a high degree of 35S-TBPS binding included the inferior colliculus, medial septal nucleus, central and paracentral nuclei of the thalamus, olfactory tubercle, zona incerta, dentate gyrus, and substantia nigra. 35S-TBPS preferentially bound to the molecular vs granular layer of the cerebellum. Omission of the preincubation markedly but variably decreased 35S-TBPS binding. The greatest regional decreases occurred in areas with a high degree of GABA synthesis. In addition, 35S-TBPS binding was inhibited to different degrees in the cell layers of the cerebellum. The addition of 1 microM GABA to the incubation medium of preincubated slices also produced variable decreases in 35S-TBPS binding to cerebellar layers. These findings support previous studies that demonstrate GABAA receptor heterogeneity. Our study confirms the suitability of 35S-TBPS for use as a ligand in autoradiography and demonstrates that the distribution of 35S-TBPS binding sites is significantly influenced by the preincubation-incubation conditions used.     

Ontogenetic changes in vasopressin binding site distribution in rat brain: an autoradiographic study

Binding sites for [3H]vasopressin were identified in brains of neonatal (days 0-23) and adult (90 day) Long-Evans and Brattleboro rats, using a technique of in vitro receptor autoradiography. Results indicate that the regional distribution of binding sites for [3H]vasopressin in the brain changes markedly during postnatal development. Binding sites in the septum proliferated slowly to attain adult distribution in the dorsal and lateral septum, while in other regions, such as the caudate, hippocampus and cingulate gyrus, intense labeling appeared early in development but disappeared by adulthood. In the amygdala, binding did not change during postnatal development. Binding sites in the septum appeared before vasopressinergic fibers are present. Also, binding was present for vasopressin in regions which have not yet been reported to receive vasopressinergic innervation. Therefore, it is proposed that the presence of binding sites for vasopressin is independent of the presence of vasopressin itself. This hypothesis is supported by labeling in the Brattleboro rat which was comparable to that in the Long-Evans.     

Iodinated-NPY binding sites: autoradiographic study in the rat brain

Several authors have described the presence of iodinated neuropeptide-Y binding sites on membranes of the mammalian CNS. In the present study we show a mapping of iodinated-NPY binding sites in the rat brain using receptor autoradiography. The sections were incubated with 125I-Bolton-Hunter coupled NPY (0.5-03 nM), in the absence or presence of 1 microM cold NPY. Some autoradiograms are studied by means of an image analyzer (VDC 501 Tesak) equipped with the host computer PDP 11 Digital, in order to enhance the contrast of the labeling. A very high density of NPY receptors is present in the limbic regions (hippocampus, amygdaloid complex, septal nuclei), in the cortex, and in some thalamic nuclei, while in some hypothalamic regions (paraventricular nucleus and median eminence) we detected a lower amount of NPY receptors. At the mesencephalic level, the substantia nigra presents a very high density of NPY receptors.     

Decreased muscarinic receptor binding in rat brain after paradoxical sleep deprivation: an autoradiographic study

Previous work demonstrated that paradoxical sleep deprivation (PSD) leads to a decrease in yawning behavior elicited by cholinergic agonists, suggesting that a downregulation of cholinergic muscarinic receptors may occur after PSD. More recent work using intracerebral injections of muscarinic agonists has suggested a critical role for M2 receptors in paradoxical sleep. In this study [³H]AF-DX 384 was used to investigate the effects of PSD on M2-type cholinergic receptors throughout the brain using quantitative autoradiography. After 96 h of paradoxical sleep deprivation, [³H]AF-DX 384 binding was generally reduced throughout the brain, and significantly so in the olfactory tubercle (−20%), n. accumbens (−23%), frontal caudate-putamen (−16%), islands of Callejas (−20%), piriform cortex (−24%), lateral (−26%) and medial (−24%) septum, anteromedial (−19%), ventrolateral (−22%), and lateral geniculate (−15%) nuclei of thalamus, deep layers of the superior colliculus (−15%), entorhinal cortex (−12%) and subiculum (−23%). [³H]AF-DX 384 binding was reduced in pontine structures, but not to a higher degree than in other brain areas. The observed downregulation of M2-type muscarinic receptors after PSD may be causally related to the previously reported decrease in cholinergically induced behaviors after PSD.     

Cerebello-olivary projections in the rat: an autoradiographic study

The cerebello-olivary projection was studied in the albino rat using conventional autoradiographic techniques. The results indicated that the cerebello-olivary projection in the rat is topographically organized in a pattern similar to other mammalian species. The anterior interpositus projects to the dorsal accessory olive, the posterior interpositus to the medial accessory olive, and the dentate to both lamellae of the principal nucleus. A point of controversy may arise, however, when one considers the fastigio-olivary projection.     

Dompamine receptors in the rat frontal cortex: an autoradiographic study.

Literature findings indicated that injection of low doses of [3H]spiperone results in a labelling of dopamine receptors in rat brain, but also in a labelling of serotonin receptors. Administration of pipamperone, a drug with serotonergic properties, to animals treated with [3H]spiperone reduced the serotonergic component of the binding and permitted an easier identification of dopamine receptor binding. At the level of Forceps Minor, there were elevated levels of receptors in the deeper layers of the cingulate cortex, in the region above the rhinal sulcus and in an area dorsal to the accumbens. This distribution is in agreement with the results of other biochemical, histochemical and electrophysiological studies.     

[H]Arginine vasopressin binding to rat brain: a homogenate and autoradiographic study

Arginine-vasopressin (AVP) has been implicated as a putative central neurotransmitter or neuromodulator in some brain functions. This study demonstrates binding of [³H]AVP to rat brain homogenates that is pH and temperature dependent, is saturable (K_d = 0.77 nM, B_max = 0.374 pmol/mg) and reversible. A number of AVP analogues competitively displaced the [³H]AVP binding, indicating that central AVP binding sites may have a resemblance to the peripheral (V₁) AVP vasopressor receptor. Homogenate binding occurred predominantly in the microsomal fraction (P₃) of the hypothalamus while in the hippocampus and septum binding was predominantly in the synaptosomal fraction (P₂). Autoradiographic methods showed displaceable [³H]AVP binding in the lateral septum, amygdala, supraoptic, paraventricular and suprachiasmatic nuclei of the hypothalamus supporting the results of homogenate binding in preparations of these regions.     

Retention of [99mTc]-d,l-HM-PAO in rat brain: an autoradiographic study

The regional cerebral distribution pattern of [99mTc]-d,l-HM-PAO in rat brain was studied by autoradiography. The regional cerebral uptake of this tracer is related to regional cerebral blood flow (rCBF); however, the ratio of retained radioactivity (determined by digital imaging techniques) in gray matter compared to white matter is lower than that reported for the blood flow ratio. Considerable inhomogeneity is observed in cortical gray matter for at least 60 min postinjection, demonstrating that minimal, if any, cerebral redistribution of this agent occurs.     

Memory consolidation and amnesia modify 5-HT6 receptors expression in rat brain: an autoradiographic study

Traditionally, the search for memory circuits has been centered on examinations of amnesic and AD patients, cerebral lesions and, neuroimaging. A complementary alternative might be the use of autoradiography with radioligands. Indeed, ex vivo autoradiographic studies offer the advantage to detect functionally active receptors altered by pharmacological tools and memory formation. Hence, herein the 5-HT(6) receptor antagonist SB-399885 and the amnesic drugs scopolamine or dizocilpine were used to manipulate memory consolidation and 5-HT(6) receptors expression was determined by using [(3)H]-SB-258585. Thus, memory consolidation was impaired in scopolamine and dizocilpine treated groups relative to control vehicle but improved it in SB-399885-treated animals. SB-399885 improved memory consolidation seems to be associated with decreased 5-HT(6) receptors expression in 15 out 17 brain areas. Scopolamine or dizocilpine decreased 5-HT(6) receptors expression in nine different brain areas and increased it in CA3 hippocampus or other eight areas, respectively. In brain areas thought to be in charge of procedural memory such basal ganglia (i.e., nucleus accumbens, caudate putamen, and fundus striate) data showed that relative to control animals amnesic groups showed diminished (scopolamine) or augmented (dizocilpine) 5-HT(6) receptor expression. SB-399885 showing improved memory displayed an intermediate expression in these same brain regions. A similar intermediate expression occurs with regard to amygdala, septum, and some cortical areas in charge of explicit memory storage. However, relative to control group amnesic and SB-399885 rats in the hippocampus, region where explicit memory is formed, showed a complex 5-HT(6) receptors expression. In conclusion, these results indicate neural circuits underlying the effects of 5-HT(6) receptor antagonists in autoshaping task and offer some general clues about cognitive processes in general.     

Ageing-related decline in adenosine A1 receptor binding in the rat brain: an autoradiographic study

The adenosine system has important neuromodulatory and neuroprotective functions in the brain. Several lines of evidence suggest that ageing is associated with major alterations in the adenosine system, which may be partially responsible for changes in sleep, mood, and cognition. In the present study, we examined adenosine A1 receptor density in the rat brain by means of quantitative autoradiography to obtain a detailed anatomical overview of the changes during ageing. A1 receptor binding was assessed in young, old, and senescent animals of 3, 24, and 30 months old, respectively. There was a clear age-dependent reduction in adenosine A1 receptors in most of the brain areas examined, but the magnitude of this reduction varied greatly among regions. Also, whereas some regions displayed a gradual decline in A1 binding sites across the three age classes, other regions showed a particularly strong decrease between the ages of 24 and 30 months. For example, whereas the hippocampus and thalamus showed a gradual decline in A1 binding, some cortical and septal regions showed a more abrupt decline after the age of 24 months. Since particularly in rats many studies have used animals at the age of 24 months or even less, the ageing-related decline in adenosine A1 signaling might have been underestimated.     

Hemicholinium-3 binding sites in rat brain: a quantitative autoradiographic study

Quantitative in vitro autoradiography was used to study the distribution of [3H]hemicholinium-3 ([3H]HC-3) binding sites in the rat brain. Regional concentrations of HC-3 binding sites were corrected for regional tissue quenching of tritium in a number of brain structures. Specific binding of 10 nM [3H]HC-3 was highest in the interpeduncular nucleus, followed by the caudate-putamen, olfactory tubercle, amygdala, and the medial and lateral habenulae. There was a high positive correlation between regional HC-3 binding and choline acetyltransferase activity in rat brain; however, a novel pattern of the distribution of cholinergic terminals in the subnuclei of the interpeduncular nucleus was discovered. The apparent Kd in the 1-5 nM range and the pharmacological specificity of the HC-3 binding site agreed with data for choline uptake and for the HC-3 binding site as determined in membrane preparations. HC-3 autoradiography appears to be a useful anatomical marker for cholinergic terminals.     

The dentatorubral projection in the rat: an autoradiographic study

The dentatorubral projection has been mapped in rats using autoradiography. Any part of lateral cerebellar nucleus (NL) projects throughout the contralateral parvocellular red nucleus (NRp) rostrocaudally; the projection is topographically organized: (1) a caudorostral shift in the NL corresponds to a dorsoventral displacement through the NRp; matching of this arrangement with the origin of rubrospinal projections is discussed; (2) only ventral parts of the NL, including the parvocellar subnucleus, project to the lateral edge of the NRp.     

Localized alterations of dopamine receptor binding in rat brain by repeated electroconvulsive shock: an autoradiographic study

The effects of repeated electroconvulsive shock (ECS) on binding parameters of D1 and D2 type dopamine (DA) receptors were investigated in different brain regions of male rats using quantitative autoradiography. D1 binding was studied with [3H]SCH 23390 as the ligand and D2 binding with [3H]spiroperidol. The distribution patterns of both D1 and D2 receptor sites were in good agreement with previously published reports. Repeated ECS induced upregulation of D1 receptors in the olfactory tubercle, the endopiriform nucleus and the substantia nigra without appreciably affecting D1 binding sites in the striatum, n. accumbens or in other brain regions containing D1 binding sites. Upregulation of D2 binding sites, after ECS, was seen in the accumbens, the olfactory tubercle, the amygdaloid nuclei, the claustrum and the endopiriform nucleus, but not in the caudate-putamen or in other brain regions containing D2 binding sites. The present finding that repeated ECS can selectively upregulate DA receptor binding sites in discrete brain areas, including limbic structures, renders important support to a large number of previous studies that demonstrated effects of repeated ECS on DA receptor function in behavioral models.     

A computerized image analysis system for quantitative analysis of cells in histological brain sections

We propose a reliable method for automatic counting of cells in brain sections labeled with different antibodies (against NeuN, parvalbumin, GABA and c-Fos) and in Nissl-staining. Images of stained sections are converted to binary images by thresholding. Clusters of 'ON pixels' (value of 1) corresponding to cell bodies are selected based on size. The parameters of the algorithm (intensity range and cluster-size) are adjusted for different methods of staining according to expert knowledge. The automatic cell counting method (ACCM) provides correct counting results, as demonstrated by a comparison of computational results with counts gained by human experimenters and with a commercially available image analysis system. On the basis of ACCM counts, small and perhaps physiologically relevant differences in the number of labeled cells can be revealed, as demonstrated here for the GABAergic system following electrical stimulation.     

Chronic buspirone treatment normalizes regional serotonin synthesis in the olfactory bulbectomized rat brain: an autoradiographic study

The effects of chronic buspirone treatments, administered by minipump at doses of 10 and 20 mg/(kg day) for 14 days, on brain 5-HT synthesis in olfactory bulbectomized (OBX) rats were evaluated. The alpha-[14C]methyl-L-tryptophan autoradiographic method was used. We compared the synthesis in the buspirone treated OBX rats (administered either 10 mg/(kg day) (OBX-10) or 20 mg/(kg day) (OBX-20)) to that of the saline treated OBX rats (OBX-SAL), and the sham operated rats (SHX) treated with saline. In addition, OBX-10 rats were compared to SHX rats treated with 10 mg/(kg day) (SHX-10) of buspirone. All treatments were carried out for 14 days. Adult Sprague-Dawley rats were used. Two weeks following the OBX or SHX procedures, the rats were assigned to the OBX-10, OBX-20, OBX-SAL, SHX-10, or SHX-SAL groups, respectively. The 5-HT synthesis rates R (pmol/(g/min)) were calculated from the trapping constant of alpha-[14C]MTrp (K*; ml/(g min)) and the plasma concentration of the plasma non-protein-bound tryptophan (Cp; pmol/ml) using the lumped constant (LC) measured previously in the rat brain. There was no significant difference in the plasma free or total tryptophan among these groups. The overall synthesis in the OBX-10 group was not statistically different from the OBX-SAL group, but it was different from the OBX-20 and SHX-SAL groups. The OBX-20 rats had an overall significant reduction in 5-HT synthesis, when compared to the OBX-SAL group, but did not differ from the SHX-SAL group, which did not differ from the SHX-10 group. These results suggest that 10 mg/(kg day) of buspirone for 14 days in the OBX rats did not produce a significant alteration in 5-HT synthesis, but 20 mg/(kg day) for 14 days resulted in an overall significant reduction in brain 5-HT synthesis. The latter treatment brought the synthesis to the level found in the sham operated rats, i.e., a normal level. These results suggest that normalization (reduction to the level found in the SHX-SAL rats) of 5-HT synthesis in the OBX requires a greater dose of buspirone (20 mg/(kg day)) than that needed to produce a desensitization of the 5-HT1A receptors in the sham operated rats (10 mg/(kg day)). This probably indicates that 5-HT1A receptors have different functionality in the OBX rats than that found in the intact or sham operated rats. Furthermore, our results support the hypothesis that 5-HT1A receptors mediate the antidepressant-like effect of 5-HT1A agonists, as the chronic 5-HT1A agonist treatment in the depression model known to be sensitive to antidepressants resulted in the normalization of 5-HT synthesis.     

Pre- and postnatal development of high-affinity [3H]nicotine binding sites in rat brain regions: an autoradiographic study.

The ontogeny of high affinity nicotinic cholinergic binding sites was studied in Long-Evans rat brain by in vitro autoradiography, using [3H]nicotine (10 nM) and cold (-)nicotine bitartrate to assess specificity. The first binding sites become detectable in spinal cord and caudal medulla oblongata at gestational day (GD) 12. Until GD 14, labelling spreads throughout lower brainstem, mesencephalon and parts of diencephalon, with higher densities in ventral areas (including the area of developing mesencephalic dopamine neurons). Matrix zones remain unlabelled. Receptor sites appear in the cerebellar anlage by GD 15, and in caudal caudate-putamen by GD 16. During development from late gestational to early postnatal stages, labelling is reduced in many lower brainstem areas and increases in forebrain, in particular in neocortex. Receptor density remains high in thalamus. In neocortex, nicotinic receptor sites are first seen in the subplate layer by GD 20. Labelling of this zone remains prominent until PN 14, when an additional band of increased receptor density is seen in cortical layers III/IV which contain high receptor levels in adulthood. At PN 27, the pattern has become similar to the adult one. The development of [3H]nicotine-binding sites in individual brain regions, with a general caudo-rostral gradient, accompanies cell differentiation and early synapse formation, e.g., in neocortex. The ontogenetic pattern differs in detail from that of muscarinic-cholinergic binding sites. The early presence of binding sites provides a basis for specific actions of nicotine on the fetal brain. As a consequence of the ontogenetic changes, different brain structures become targets for the action of this drug at different stages of development.     

A three-dimensional statistical analysis for CBF activation studies in human brain.

Many studies of brain function with positron emission tomography (PET) involve the interpretation of a subtracted PET image, usually the difference between two images under baseline and stimulation conditions. The purpose of these studies is to see which areas of the brain are activated by the stimulation condition. In many cognitive studies, the activation is so slight that the experiment must be repeated on several subjects and the subtracted images are averaged to improve the signal-to-noise ratio. The averaged image is then standardized to have unit variance and then searched for local maxima. The main problem facing investigators is which of these local maxima are statistically significant. We describe a simple method for determining an approximate p value for the global maximum based on the theory of Gaussian random fields. The p value is proportional to the volume searched divided by the product of the full widths at half-maximum of the image reconstruction process or number of resolution elements. Rather than working with local maxima, our method focuses on the Euler characteristic of the set of voxels with a value larger than a given threshold. The Euler characteristic depends only on the topology of the regions of high activation, irrespective of their shape. For large threshold values this is approximately the same as the number of isolated regions of activation above the threshold. We can thus not only determine if any activation has taken place, but we can also estimate how many isolated regions of activation are present.     

Dopamine receptors and learned helplessness in the rat: an autoradiographic study

(1) Disturbances of mesolimbic and mesocortical dopamine (DA) function have been implicated in the pathophysiology of several psychiatric disorders, including major depressive disorder. (2) Utilizing the learned helplessness (LH) animal model of clinical depression and quantitative autoradiography, the authors studied the densities of D1 and dopamine-2-like receptors (D2-like receptors) in medial prefrontal cortex, septum, nucleus accumbens and caudate nucleus in rats that received inescapable stress and were subsequently tested for LH behavior. (3) Dopamine-1 receptor (D1 receptor) densities were significantly higher in the core and shell of the nucleus accumbens and in the medial caudate nucleus of rats that did not become helpless after stress, compared to rats that developed LH. (4) Densities of D2-like receptors were significantly lower in the core of the nucleus accumbens in both the LH and the nonhelpless (NH) rats compared to controls. Densities of D2-like receptors were also lower in the medial and lateral caudate nuclei in LH rats compared to the other groups. (5) Increased D1 receptor densities in NH rats in the nucleus accumbens may be associated with an adaptive or protective role of this brain region in the prevention of escape deficits after exposure to inescapable stress. (6) Decreased D2-like receptor densities in the caudate nucleus in helpless rats may reflect a motor deficit associated with LH behavior, while decreases of D2-like receptor densities in the core of the nucleus accumbens may reflect a generalized effect of exposure to inescapable stress. (7) This study highlights the importance of the mesolimbic/nigrostriatal dopaminergic systems in mediating behavioral responses to inescapable stress.