Dendritic thickness: a morphometric parameter to classify mouse retinal ganglion cells

To study the dendritic morphology of retinal ganglion cells in wild-type mice we intracellularly injected these cells with Lucifer yellow in an in vitro preparation of the retina. Subsequently, quantified values of dendritic thickness, number of branching points and level of stratification of 73 Lucifer yellow-filled ganglion cells were analyzed by statistical methods, resulting in a classification into 9 groups. The variables dendritic thickness, number of branching points per cell and level of stratification were independent of each other. Number of branching points and level of stratification were independent of eccentricity, whereas dendritic thickness was positively dependent (r = 0.37) on it. The frequency distribution of dendritic thickness tended to be multimodal, indicating the presence of at least two cell populations composed of neurons with dendritic diameters either smaller or larger than 1.8 æm ("thin" or "thick" dendrites, respectively). Three cells (4.5 percent) were bistratified, having thick dendrites, and the others (95.5 percent) were monostratified. Using k-means cluster analysis, monostratified cells with either thin or thick dendrites were further subdivided according to level of stratification and number of branching points: cells with thin dendrites were divided into 2 groups with outer stratification (0-40 percent) and 2 groups with inner (50-100 percent) stratification, whereas cells with thick dendrites were divided into one group with outer and 3 groups with inner stratification. We postulate, that one group of cells with thin dendrites resembles cat ß-cells, whereas one group of cells with thick dendrites includes cells that resemble cat a-cells.

Dendritic thickness: a morphometric parameter to classify mouse retinal ganglion cells.

To study the dendritic morphology of retinal ganglion cells in wild-type mice we intracellularly injected these cells with Lucifer yellow in an in vitro preparation of the retina. Subsequently, quantified values of dendritic thickness, number of branching points and level of stratification of 73 Lucifer yellow-filled ganglion cells were analyzed by statistical methods, resulting in a classification into 9 groups. The variables dendritic thickness, number of branching points per cell and level of stratification were independent of each other. Number of branching points and level of stratification were independent of eccentricity, whereas dendritic thickness was positively dependent (r = 0.37) on it. The frequency distribution of dendritic thickness tended to be multimodal, indicating the presence of at least two cell populations composed of neurons with dendritic diameters either smaller or larger than 1.8 microm ("thin" or "thick" dendrites, respectively). Three cells (4.5%) were bistratified, having thick dendrites, and the others (95.5%) were monostratified. Using k-means cluster analysis, monostratified cells with either thin or thick dendrites were further subdivided according to level of stratification and number of branching points: cells with thin dendrites were divided into 2 groups with outer stratification (0-40%) and 2 groups with inner (50-100%) stratification, whereas cells with thick dendrites were divided into one group with outer and 3 groups with inner stratification. We postulate, that one group of cells with thin dendrites resembles cat beta-cells, whereas one group of cells with thick dendrites includes cells that resemble cat alpha-cells.

Local application of L- threo-hydroxyaspartate and malonate in rats in vivo induces rigidity and damages neurons of the substantia nigra, pars compacta.

In order to study neuronal death in Parkinson's disease, neurons of the substantia nigra, pars compacta in rats were exposed to elevated levels of glutamate and decreased levels of energy in vivo and consequences for behavior and neuronal morphology were studied. Thus, repeated local injections (9x) of the glutamate uptake inhibitor L- threo-hydroxyaspartate (L-THA; 833 microM in 0.3 microl) in the presence or absence of the succinate dehydrogenase inhibitor malonate (25 mM in 0.3 microl) were applied during three weeks. 24 h after injection, rigidity and catalepsy were measured, as well as, at the end of the three week period, locomotion, rearing and exploratory behavior. Thereafter, the cytoarchitecture of the substantia nigra, pars compacta of the brains of these rats was described. The L-THA plus malonate injected rats did not differ in their behavior from carrier injected rats, except for rigidity: their scores were higher than that of carrier and L-THA injected rats (P < 0.05), while L-THA injected rats did not differ from carrier injected controls. Observations on cresyl violet sections revealed, that, although many neurons with a shrunken nucleolus and faintly stained cytoplasm were present in both L-THA and L-THA plus malonate treated rats, the ventral edge of the substantia nigra, pars compacta containing modified cells was longer in L-THA plus malonate than in L-THA injected rats (P < 0.05). This indicates, that a minimum amount of damage to neurons in the ventral part of the substantia nigra, pars compacta might be required for the expression of rigidity.

Soma size of substantia nigra neurons increases after a prenatal neocortical lesion in cats.

Seeking an explanation for an increase in volume of the caudate nucleus in adult cats that had sustained a fetal unilateral neocortical lesion, we investigated possible morphological changes in the reciprocally interconnected substantia nigra. In fetal-lesioned cats the cross-sectional area of neuronal somata in substantia nigra, pars reticulata was 33% larger than in control cats (P<0.05), while in pars compacta there was a marked tendency to an increase (25%, P<0.06). This size increase might have caused the survival of a larger number of caudate nucleus neurons during development, and thus contributed to the reported increase in caudate nucleus volume.

Differences in D2 dopamine receptor binding in the neostriatum between cats hemidecorticated neonatally or in adulthood.

In order to study differences in response to neocortical injury sustained at different ages at the neurotransmitter level, we examined the density in D2 dopamine receptors in the neostriatum of cats hemidecorticated neonatally (N = 4) or in adulthood (N = 4), as well as in intact brains (N = 6). Receptor densities were measured using quantitative autoradiography and [3H]-spiperone binding in 12 regions of the neostriatum and nucleus accumbens septi. We found that the anterior lateral caudate nucleus on both sides of the brain contained a higher D2 receptor density in neonatal-lesioned as compared to adult-lesioned brains. Ipsilateral to the lesion, the increase was 101% (P < 0.05) and contralaterally it amounted to 77% (P < 0.05). Moreover, this region of the ipsilateral caudate nucleus of neonatal-lesioned cats tended to be more densely labeled than that of intact brain by 58% (P < 0.1). D2 receptor densities in adult-lesioned cats did not differ from that of intact controls. Comparison of these data with those of a former morphological study using the same animals suggested that this bilateral elevation of D2 receptor density in neonatally lesioned brains represents a higher mean density of binding sites per neuron. The elevation in the neonatal-lesioned cats might be a response of the striatum to neuroplastic changes in the striatal neuropil, including the corticostriatal afferents, since such changes are different in neonatal- as compared to adult-lesioned cats.

The role of NMDA receptors in the slow neuronal degeneration of Parkinson's disease.

Parkinson's disease is a disorder, in which neurons of various neuronal systems degenerate. Furthermore, in such degenerating neurons, the cytoskeleton seems to be affected. In this respect, Parkinson's disease resembles Alzheimer's disease. Since it has been shown, that elevated levels of intracellular calcium can disrupt the cytoskeleton and that the stimulation of glutamate (NMDA) receptors can cause high intracellular concentrations of calcium, it has been suggested, that the stimulation of glutamate receptors plays a role in the slow degeneration in Alzheimer's and Parkinson's disease. In case of the degeneration of the dopaminergic nigrostriatal system in Parkinson's disease, neurons that contain calcium binding protein appear to be less vulnerable than the neurons that lack it, suggesting that calcium binding protein might protect these neurons from degeneration by preventing that cytosolic calcium concentrations increase excessively. And, since there is in the nigrostriatal system a glutamatergic afferent pathway (the prefrontonigral projection) and since dopaminergic nigrostriatal neurons contain postsynaptic NMDA receptors, glutamatergic excitation may play a role in the degeneration of the nigrostriatal system in Parkinson's disease. If so, it may be possible to protect the neurodegeneration of these dopaminergic neurons by NMDA receptor antagonists.

Injection of dye into neurones in rat and human post-mortem brain, in combination with acetylcholinesterase histochemistry: permanent preparations.

We describe a protocol for the intracellular injection of dye into neurones in thick sections of fixed, post-mortem rat and human brain tissue. To render the sections with the intracellularly injected neurones permanent, they are sectioned again, and the resulting subsections are either immunocytochemically treated or stained histochemically for acetylcholinesterase (AChE) activity. The resultant preparations can be stored at room temperature for prolonged periods. Background staining produced by accumulation of erythrocytes in blood vessels is greatly reduced or virtually eliminated by exposure of the sections to ultraviolet radiation prior to the intracellular injection. The pattern of AChE staining is not affected by this procedure. This ability to stain sections according to a histochemical AChE procedure after the intracellular injection of dyes into striatal neurons opens the possibility to study the relationship of neuronal dendritic trees with the striosome/matrix compartmental boundaries in post-mortem (human) brain tissue of Huntington's disease patients.

The effect of neocortical lesions on the number of cells in neonatal or adult feline caudate nucleus: comparison to fetal lesions.

After a unilateral resection of the frontal cortex in fetal cats the volume of the caudate nucleus increases while the packing density of neuronal and glial cells does not change. In the present report we address the questions of whether a similar lesion sustained neonatally or a more extensive neodecortication sustained neonatally or in adulthood may have the same unusual effect. Stereological methods were used to determine bilaterally the volume of the caudate nucleus as well as to estimate the total number and packing density of neurons and glial cells in the caudate nucleus ipsilateral to the lesion. Comparisons between each of three experimental groups and intact animals were made at a time when all animals were young adults. In cats with a unilateral frontal cortical lesion performed between postnatal days 8 and 14, none of the measured parameters changed significantly compared to intact controls. In cats with removal of the entire left neocortex in adulthood, the ipsilateral caudate nucleus volume decreased by 18.1% and by 21.5% relative to intact and to neonatal hemidecorticated cats respectively (P < 0.05), with no change in the contralateral caudate. In the ipsilateral caudate the total number of neurons decreased by 21.8% (P < 0.05) compared to controls while the number of glial cells did not change significantly. In the same caudate the packing density of neurons did not change significantly (except for a 17.1% decrease, P < 0.05, relative to frontal-lesioned cats) while that of glial cells increased by 19.9% and by 24.7% compared to intact and neonatal neodecorticated cats respectively (P < 0.05). In adult cats in which a similar hemineodecortication was performed between postnatal days 8 and 13, the only significant changes were a 25.8% (P < 0.05) and a 30.6% (P < 0.05) decrease in neuron packing density compared to intact and frontal-lesioned cats, respectively. In summary, a restricted unilateral neocortical resection in neonatal cats did not induce any morphological changes in the caudate nucleus that we could detect with the methods employed. In contrast, an extensive neodecortication sustained in adulthood produced ipsilateral caudate shrinkage with substantial neuron loss and increase in packing density of glial cells, while a similar lesion but sustained neonatally only altered substantially the packing density of glial cells (decreased). Therefore, we concluded that (i) the caudate nucleus hypertrophy which we reported after a unilateral discrete cortical removal during the prenatal period is a unique phenomenon which is peculiar to the cat brain during the last third of gestation; (ii) the caudate nucleus changes seen in the cats with hemineodecortication in adulthood are degenerative in nature and closely resemble those which we reported for other subcortical nuclei following a similar lesion; and (iii) the animals with neonatal hemidecortication are relatively spared from these degenerative effects. Overall, these results indicate that, as for other structures, the morphological changes of the caudate nucleus following neocortical damage depend on the maturational state of the brain at the time of the injury and on the size of the lesion, and support the notion that the consequences of cerebral cortex lesions upon subcortical brain nuclei are of a different nature when sustained in prenatal as compared to postnatal cats.

Innervation of the caudate nucleus, thalamus and red nucleus by the remaining sensorimotor cortex in cats with fetal or neonatal unilateral frontal cortex removal.

We studied the projections to the caudate nuclei, thalami and red nuclei from the remaining sensorimotor cortex in adult cats that had sustained a unilateral frontal cortex resection prenatally or neonatally. Four cats had the lesion at age E 50-55 and six animals sustained the ablation at age P 8-14 (seven cats were intact controls). All cats grew to young adulthood and then received injections of tritiated leucine-proline in the remaining sensorimotor cortex. Injection sites and axon terminal fields were reconstructed using autoradiography-processed tissue. In all cats the label filled a similar extent of the right pericruciate cortex. Terminal field densities in the subcortical nuclei were estimated using computer-based video software. Three medial-lateral sectors at five coronal levels were examined in the caudate nucleus. Three nuclear groups were analyzed in the thalamus (intralaminary, ventralis lateralis and ventrobasal complex). For the red nucleus, the four quadrants were examined at four coronal levels. The main goal of the study was to assess possible changes in the cortical innervation of the nuclei ipsilateral to the lesion. Therefore, the mean particle counts per nucleus (and per area or sector of nuclei) and per animal group were used to calculate percentage values for the decussated (crossed, or contralateral to the injection site) as a function of the non-decussated (uncrossed, or ipsilateral to the injection site) innervation. The percentage values for the crossed projections were: (a) for the entire caudate nucleus, 61.3% for the intact. 56.7% for the fetal-lesioned and 42.7% for the neonatal-lesioned cats, with no statistical differences between groups; (b) for the thalamus the proportion of crossed projections was minimal fluctuating between a low 0.06-0.16% for the nucleus ventralis lateralis and a high of 2.01-3.46% for the intralaminary nuclei, with the highest values belonging to the lesioned groups but with no significant differences between groups: (c) for the entire red nucleus, 1.98%, 12.74% (P < 0.05) and 6.76% for the intact, fetal- and neonatal-lesioned cats respectively. In the lesioned cats, the topography of the distribution of the axon terminals was bilaterally the same as in the controls. In conclusion, only the red nucleus of the frontal-lesioned cats showed an increased crossed innervation from the remaining sensorimotor cortex but this was relatively weak and statistically significant only for the fetal-lesioned animals. These results as well as the literature suggest that: (a) the crossed corticorubral projections in fetal cats may represent true reinnervation (i.e., newly originated, no preexisting terminals); (b) the relative paucity of the crossed projections in the present cats as compared to the extensive reorganization of subcortical terminals seen after cerebral hemispherectomy (our original postnatal lesion model) may be due to the much smaller size of the present cortical lesion which presumably induced only a limited amount of subcortical nuclear deafferentation.

Few neocortial and thalamic morphological changes after a neonatal frontal cortical ablation contrast with the effects of a similar lesion in fetal cats.

To further understand the neuroanatomical consequences following perinatal brain injury, quantitative morphometric analysis was performed on the brain of cats receiving a unilateral frontal cortical ablation between postnatal days (P) 9 and P 14 and intact control cats. In all cats, the volume of the neocortex and thalamus was measured bilaterally and that of the thalamic ventrobasal complex (VBc) was measured ipsilaterally. In addition, using stereology, the neuronal and glial (presumably) cell packing densities (CPD) and the total number of neurons and glial cells (TCN) were measured in the ipsilateral VBc. The neuronal and glial cell cross-sectional areas (CSA) were also measured in the ipsilateral VBc. The mean ipsilateral and contralateral neocortex volumes were similar between the two animal groups. There was a statistically significant 14% and 13% reduction in mean ipsilateral and contralateral thalamic volumes, respectively for the lesioned animals, while the VBc shrank by 16% relative to intact controls. The mean neuronal and glial CPD were similar between the two groups. The mean neuronal TCN was reduced by 10% in the neonatal-lesioned cats, while the mean glial TCN was reduced by 31% in the same animals, however neither value reached significance. Lastly, the mean CSA of neurons and glial cells showed a tendency to be smaller in the lesioned cats by 8% and 9%, respectively. These results: (a) indicate that the neonatal lesion caused only minor morphological brain alterations and this sharply contrast with the marked changes previously reported in cats with a similar lesion sustained prenatally; (b) suggest that the enhanced behavioral recovery and/or sparing reported for the present cats compared to fetal-lesioned animals is at least partially due to the morphological sparing reported here; (c) together with previous findings in fetal cats, support the hypothesis that the morphological changes after a neonatal neocortical lesion are qualitatively different and may depend on different mechanisms as compared to those occurring after similar damage sustained prenatally.

Morphological changes in the thalamus and neocortex of the cat brain after a restricted unilateral fetal neocortical lesion.

In order to study the response of the brain to injury during early development, the neocortex of ten fetal kittens was lesioned at age E43-48, in either the frontal (n = 8) or parieto-occipital (n = 2) areas. The thalamus and neocortex of the lesioned animals were analyzed using quantitative morphometry and compared to intact control cats (n = 10). Ipsilaterally, the volumes of the remaining neocortex and of the thalamus were 26.5% and 25.7% smaller, respectively (P < 0.05). Contralaterally, the neocortex did not change in volume, whereas the thalamus tended to be smaller by a mean of 11.1%. Ipsilaterally, in all four thalamic nuclei studied, the neuronal and glial cell packing densities (NCPD and GCPD) and the cross sectional area of neuronal somata did not differ between lesioned and intact animals except for the principal ventromedial nucleus, where the GCPD was significantly lower (P < 0.05) in lesioned animals. Contralaterally, the NCPD and GCPD did not show any differences between groups, except for the principal ventromedial nucleus, in which the GCPD was lower in lesioned cats (P < 0.05). Furthermore, in the contralateral basal ventromedial nucleus, the cross sectional area of the neuronal somata was smaller in lesioned than in intact animals (P < 0.01). These results indicate loss of neurons and glia in the ipsilateral thalamus and probably in the neocortex. Since, at the time of the cortical resection, transient reciprocal thalamosubplate connections have been established in the cat, the lesion-induced deprivation of subplate target neurons and cortical inputs probably precluded the survival of a substantial number of developing thalamic neurons. In the cortex the hypothetical loss of neurons may, at least partly, be attributed to lesion-induced elimination of target neurons before establishment of corticocortical connections.

Increase in size of the caudate nucleus of the cat after a prenatal neocortical lesion.

In order to study the influence of prenatal developmental factors upon reaction of the brain to injury, fetal kittens (E43-48) were lesioned in the frontal or parietal cortex unilaterally and maintained into young adulthood. The animals were sacrificed by perfusion with an aldehyde fixative or by an overdose of pentobarbital. Frozen sections were cut and stained with thionin. These sections were used for calculation of caudate nuclei volumes and for measurements of neuronal and glial cell packing density and neuronal cell body size. Island and matrix compartments were sampled separately. We found that the volume of the ipsilateral caudate nucleus had significantly increased as compared to (a) the volume of the corresponding nucleus in intact cats (mean, 15%) and (b) the nucleus contralateral to the lesion (mean, 7.6%). The latter nucleus also tended to a volume increase (mean, 8.1%). The cytoarchitecture of the caudate nuclei was essentially unchanged with two exceptions. The neuronal cell packing density in the matrix compartment of the contralateral side was decreased (mean, 14.9%) while the size of neuronal cell bodies in the island compartment of the nucleus ipsilateral to the lesion was smaller (mean, 5%) relative to controls. These findings suggest that the number of neurons in the caudate of lesioned animals was larger than in intact controls, particularly on the lesioned side of the brain. This might be due to a reduction of naturally occurring cell death during development.

Loss of dopamine receptors in the olfactory bulb of patients with Alzheimer's disease.

The presence and localization of dopamine D2 receptors was studied by means of in vitro autoradiography with [3H]N-n-propylnorapomorphine in olfactory bulbs obtained postmortem from Alzheimer patients and age-matched controls. It appeared that, in 5 age-matched controls, the greatest density of dopamine D2 receptors was found in the glomerular layer of the bulb. In 6 of 7 Alzheimer patients, the labeling of the glomerular layer was decreased so that glomerular and granular layer did not differ in labeling density. Tangles, as revealed by Thioflavin S staining, were present in all different layers of the bulbus in Alzheimer patients. Observation of Nissl-stained preparations revealed that mitral cell bodies in bulbs of these patients were not present. Since mitral cells are projection neurons with targets in the entorhinal and piriform cortex, the observation of loss of these cells supports the hypothesis of early involvement of the olfactory system in Alzheimer's disease and the spread from the olfactory mucosa and bulb to the cerebral cortex and hippocampus via degeneration of interconnecting neurons. Moreover, in vivo detection of bulbar dopamine receptors might in the future provide a diagnostic tool for the early detection of senile dementia of the Alzheimer type.

Distribution of dopamine D-2 receptors in the rat striatal complex and its comparison with acetylcholinesterase.

The distribution of D-2 dopamine receptors in the rat striatal complex was studied with autoradiography after specific in vivo labeling with the dopamine agonist [3H]N-n-propylnorapomorphine and subsequent irreversible fixation. This labeling technique allows the visualization of D-2 receptors at the cellular level by light microscopic emulsion autoradiography. During the preparation of emulsion autoradiograms, the recovery of the label was 75%, the specific and the aspecific label being equally affected. The distribution of label before and after the loss of radioactive label occurred, did not show differences. In rat neostriatum, dopamine D-2 receptors are not homogeneously distributed: in the caudate-putamen the density is laterally higher than medially. Moreover, there exists a mosaic-like pattern of receptor density. In the ventral striatum, comprising the fundus striati, nucleus accumbens septi and olfactory tubercle, the receptor density is lower than in the caudate-putamen, except for the core regions in the islands of Calleja and the rim of these islands, which contain high (as high as the lateral caudate-putamen) and a moderate density of receptors, respectively. In caudate-putamen and lateral nucleus accumbens it appeared that the intensity of acetylcholinesterase staining parallels more or less the distribution of dopamine D-2 receptors. In medial nucleus accumbens and in olfactory tubercle, the high intensity of acetylcholinesterase is not paralleled by a high D-2 receptor labeling density. This receptor labeling density does not seem to be matched by differences in densities of medium-sized neuronal cell bodies.

Regional differences in reappearance of D2-dopamine receptors in the rat caudate-putamen complex after irreversible inactivation.

The reappearance of D2-receptors in the striatum of the rat was studied by autoradiography after in vivo labeling with [3H]N-n-propylnorapomorphine ([3H]NPA) at various time intervals after the inactivation of dopamine receptors by intraperitoneal administration of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). Within two days after inactivation the labeling had decreased to 18% of controls. Thereafter, the label reappeared and after 8 days or more reached levels of 80% of that of untreated controls. Autography showed that 4 h after EEDQ treatment no preferential labeling of the striatum can be seen. Five days after EEDQ a slight difference in labeling density between the medial and lateral striatum was detected, whereas after 18 days a prominent lateromedial gradient in silver grain density was seen, resembling the gradient seen without EEDQ treatment. This silver grain gradient is not paralleled by the density of medium-sized neuronal cell bodies. This suggests a difference in synthesis rate of receptors either in other cells than the medium-sized neuron or, alternatively, in otherwise indistinguishable medium-sized neurons. Five days after EEDQ treatment, clusters of silver grains in the lateral striatum were seen. These clusters have a diameter of 150-400 microns and are separated from each other at 200-500 microns. Each cluster may represent newly synthesized receptors of a single neuron (e.g. cholinergic or somatostatinergic interneuron).

Synaptic and non-synaptic striatal dopamine D2 receptors: possible implications in normal and pathological behaviour.

Various levels of organisation in the central nervous system can be distinguished, ranging from the molecular, the cellular, the multicellular and the neuronal system level. The relationship between receptor function and behaviour is focussed to the dopamine D2 type receptor of the striatal complex in relation to extrapyramidal and limbic systems. In the striatal complex a striosomal and a matrix compartment can be distinguished. The matrix compartment can be considered as a part of the extrapyramidal system and is innervated by the motor cortex and by the dopaminergic neurons of the ventral tegmental, the dorsal substantia nigra and the retrorubral area. This compartment has a relatively high density of D2 receptors. The striosomes are innervated by e.g. the prelimbic cortex and dopamine neurones of the ventral part of the substantia nigra; here the density of D2 receptors are lower. Under normal conditions most of the D2 receptors are occupied by endogenous dopamine, and postsynaptic (e.g. cholinergic) function is therefore sensitive to antagonists; e.g. antipsychotics. Exposure to drugs such as amphetamine produces a substantial overflow of dopamine from nerve terminals leading to the activation of remote dopamine receptors, that may belong to the system that normally is not influenced by these nerve terminals (defined here as extra synaptic receptor activation). A loss of the normal spatial-temporal relationships may also occur during L-DOPA therapy in Parkinson's disease. In this illness, due to degeneration of dopaminergic innervation, several dopamine receptors have become non-synaptic. In these states of intoxication the normal spatial/temporal organization is lost and such a loss may contribute to behavioural impairments.

A mosaic-like distribution of dopamine receptors in rat neostriatum and its relationship to striosomes.

The distribution of dopamine receptors in rat neostriatum was determined by means of light microscopic autoradiography of in vivo labeled binding sites for [3H]N-n-propylnorapomorphine and compared with the distribution of acetylcholinesterase histochemical staining. The distribution of dopamine receptors was non-homogeneous and patches of low receptor density were in register with acetylcholinesterase-poor striosomes. This suggests that the distribution pattern of dopamine receptors is specifically related to various input systems.

In vivo labeling of dopamine receptors: light microscopic localization at the cellular level by means of dipping autoradiography with the agonist (3H)N-n-propylnorapomorphine.

A convenient method is described for light microscopic autoradiography at the cellular level for the visualization of in vivo labeled dopamine receptors. (3H)N-n-propylnorapomorphine [(3H)NPA] is administered to rats under conditions that are known to give specific and saturable accumulation in the striatum. Seventy minutes after intravenous administration, the brain is rapidly frozen and cut on a cryostate microtome. The sections are treated with formaldehyde vapor, defatted for 1 hour in xylene and dipped in liquid nuclear emulsion, exposed for 2 weeks and developed. Autoradiograms obtained in this way show a high silver grain density over the striatum and a low density over adjacent external capsule and neocortex. There was a sharp delineated boundary between striatum and adjacent external capsule. Since low energy radiation can be quantified in liquid emulsion autoradiograms, we counted silver grains in a number of regions. The distribution of silver grains appeared to be identical with the distribution of radioactive label after similar in vivo administration of (3H)NPA in other studies.

Organization of the striatum: collateralization of its efferent axons.

The anatomical structure of the basal ganglia indicates that the input from the cerebral cortex is funnelled through the striatum to the globus pallidus and substantia nigra. This structure implies integration of the information as it is transferred through the basal ganglia. In order to investigate this integration, we studied the collateralization of striatal efferents to the globus pallidus and the substantia nigra-ventral tegmental area. Retrogradely transported fluorescent tracers were injected into the target areas of striatal efferents. Nuclear yellow or propidium iodide was injected into the substantia nigra-ventral tegmental area (SN-VTA) and 4-acetamido, 4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) into the globus pallidus (GP) of adult albino rats. SITS was chosen for the pallidal injections because it is not taken up by fibers-of-passage. The pressure injections resulted in large injection sites which covered the majority of each efferent target area, and as a result retrogradely labeled cell bodies were found throughout the entire extent of the striatum. Cell bodies double-labeled with both dyes were found intermingled with single-labeled cell bodies. In rats injected with propidium iodide in the SN-VTA and SITS in the GP, 70% of all neurons (as revealed by Nissl staining) were labeled. Of these labeled cells, 40% were double labeled, 20% contained only SITS and 40% contained only propidium iodide. Thus a substantial number of the striatal neurons that project to the SN-VTA also possess collateral axons to the GP. Some striatal neurons appear to project to only the SN-VTA or only to the GP. The cells projecting to only one of these striatal target regions tend to cluster together in patches. The organizational pattern of these patches does not seem to coincide in any simple way with the mosaic pattern of striatal opiate receptors, nor with the previously described mosaic pattern of striatal afferents and various neurotransmitter substances.

Simultaneous ultrastructural localization of cholecystokinin- and tyrosine hydroxylase-like immunoreactivity in nerve fibers of the rat nucleus accumbens.

Ultrathin frozen sections were used to study the localization of cholecystokinin (CCK) in dopaminergic systems in the rat nucleus accumbens. Antibodies against CCK and tyrosine hydroxylase (TH), a synthetic enzyme of dopamine, were differentially visualized using protein A conjugated to colloidal gold particles of different sizes. Nerve processes were observed to be immunocytochemically labelled for either CCK or TH but also in some cases for both CCK and TH. CCK-like immunoreactivity was localized in vesicles with a diameter of 70-160 nm, whereas TH-like immunoreactivity was primarily localized in the axoplasm. Most of the double-labelled nerve processes did not show pre- or postsynaptic specializations and most likely represent preterminal elements.