Parvalbumin and calbindin D-28K immunoreactive neurons in area MT of rhesus monkey

The chemical characteristics of the neurons of the motion sensitive visual area, area MT, remain to be established. We studied the distribution pattern of two calcium binding proteins, parvalbumin (PV) and calbindin D28K (CB) in this area, using specific monoclonal antibodies and the peroxidase-antiperoxidase (PAP) immunohistochemical technique. Aldehyde fixed 30-micron-thick cryostat sections from area MT of five animals were processed free floating for immunohistochemical staining. Besides studying the morphological characteristics of PV and CB positive neurons, quantitative analysis was carried out to determine their (1) perikaryal area (Pa) and diameter, (2) numerical densities (NV)/mm3 cortical tissue, (3) absolute number (NC) in a column of cortex under 1 mm2 cortical surface along with (4) layerwise absolute number (NL) under 1 mm2 cortical surface and (5) laminar percentage distribution of immunoreactive (IR) neurons. Quantitative analysis was carried out using a Leica QMC 500 image analysis system connected to a DMRE microscope. The results showed that both types of IR neurons were localized to all cortical layers except layer I. The PV +ve neurons were equidistributed between the supra- and infragranular layers, with the highest percentage being present in layer III (45%) followed by layer V (21%). The CB +ve neurons, on the other hand, were predominantly localized in supragranular layers, with the highest percentage being in layer III (54%) and the next highest percentage in layer II (18%). The average Pa and diameter of PV +ve neurons were found to be 96.90 +/- 28.43 micron 2 and 11.01 +/- 1.61 microns respectively. The CB +ve neurons were significantly smaller in size than the PV +ve neurons, with average Pa and diameter of the former being 92.23 +/- 26.18 micron 2 and 10.39 +/- 1.23 microns respectively. The NV for PV and CB +ve neurons showed ranges of 3157-3894 and 2303-2585, with means of 3347 +/- 285 (+/- SD) and 3436 +/- 100 respectively. The values for NC showed ranges of 5230-5444 and 4020-4268 with means of 5378 +/- 85 and 4167 +/- 95 for PV and CB neurons respectively. Variations in size together with the differential distribution of these neurons in the cortical layers may indicate their involvement in different functional circuitaries.     

The limbic zone of the rabbit and rat claustrum: a study of the claustrocingulate connections based on the retrograde axonal transport of fluorescent tracers

The claustrum is a subcortical structure lying under the insular and piriform cortices, whose function is still not clear. Although data exist on connections of the claustrum and the limbic cortex, the topography of the limbic zone in the rabbit and rat claustrum has not been studied extensively. The study was performed on 17 adult Wistar rats and 12 New Zealand rabbits. Two percent water solutions of fluorescent retrograde tracers fast blue and nuclear yellow were injected into the various regions of the limbic cortex. The limbic zone is localized throughout the whole rostrocaudal extent of the claustrum, mainly in its ventromedial portion lying close to the external capsule. Although this zone of the claustrum is localized similarly in both rat and rabbit, some differences between these two species exist. In the rat, neurons projecting to all limbic areas are localized mainly in the anterior and central parts of the claustrum, whereas in the rabbit, the majority of the neurons projecting to the cingulate cortex are present in the anterior and central parts of this structure, while neurons sending axons to the retrosplenial cortex are localized in the central and posterior parts. In both species, double-labeling study showed that neurons projecting to various limbic regions are intermingled and that neurons sending axons into two different limbic regions are seen only occasionally. Our findings give support to the role of the claustrum in integrating information between different areas of the cerebral cortex and the limbic system. Accepted: 11 June 1999     

Ontogeny of the calcium binding protein calbindin D-28k in the rat nervous system

Summary Calbindin D-28k immunoreactivity appeared at embryonal day 14 (E14) in the central nervous system as well as in the sensory organs and at E15 in the peripheral nervous system of the rat. At E14 the infundibular process of the diencephalon, cells of the posterior hypothalamus and of the dorsal thalamus were the only structures strongly immunostained in the brain, whereas neurons of the basal plate of the spinal cord, medulla oblongata and of the out-ermost layer of the cerebral cortex were only faintly labeled. Calbindin positive cerebellar Purkinje cells could be discerned at E15 together with a few cells in the hippocampus and in ganglia of the cranial nerves. At E19 various mesencephalic and metencephalic structures, spinal ganglion cells and basal ganglia displayed calbindin immunoreactive cells. The adult pattern of calbindin immunoreactivity (Garcia Segura et al. 1984) was reached before birth in most brain regions. In general, cells which displayed calbindin during brain development were also calbindin positive in the adult animal. Exceptions to this rule were cells of the deep nuclei of the cerebellum and non-neuronal cells which transiently expressed calbindin during development. Calbindin appeared in a given brain region almost invariably 1 or 2 days after the cessation of cell division and the beginning of neuronal migration and extension of neuronal processes. The calcium binding protein calbindin might influence these Ca²⁺-dependent processes.Abbreviations A Axon - ac anterior commissure - Acq Aqueductus cerebri - AH Adenohypophysis - AMY Amygdala - aV anterior vermis - BG Basal Ganglia - BO Bulbus olfactorius - BPG basal pontine grey - C Cortex - CA Crista ampullaris - cer Cerebellum - CO otic cyst - CP choroid plexus - CPT Caudatoputamen - DCN deep cerebellar nuclei - DT dorsal thalamus - E foregut epithelium - ec external capsule - eml external medullary lamina - ENP entopeduncular nucleus - EP Ependym - ET Epithalamus - EY eye - F Fimbria - fo Fornix - fr Fasciculus retroflexus - GP Globus pallidus - gr granular laver of the cerebellum - Gsp ganglion spirale cochlea - HI Hippocampus - HYP Hypothalamus - I hippocampal interneurons - ic internal capsule - IE inner ear - IH inner hair cells of the cochlea - IO inferior olive - IS internal sulcus cells of the cochlea - LD laterodorsal thalamus - LV lateral ventricle - MB mamillary body - MH medial habenular nucleus - mol molecular layer of the cerebellum - mt mamillo-thalamic tract - mtt mamillo-tegmental tract - N nose - n olfactory nerve - NBM Nucleus basalis of Meynert - NE nose epithelium - NH Neurohypophysis - NRT Nucleus reticularis tegmenti pontis - ON olfactory nuclei - OR optic recess - PC posterior commissure - PG Epiphysis - PI inferior cerebellar peduncle - PS superior cerebellar peduncle - PU Purkinje cell - PV paraventricular hypothalamic nucleus - pV posterior vermis - PVP paraventricular thalamic nucleus - PY pyramidal cells of the hippocampus - RA Raphe nuclei - RE Nucleus reuniens - RH Nucleus rhomboideus - RN reticular nucleus - RP Rathke's pouch - sc spinal cord - SG substantia gelatinosa Rolandi - slm Stratum-lacunosum molecular - sm Stria medullaris - SN Substantia nigra - SO supraoptic nucleus - Sol Nucleus of the solitary tract - Tect Tectum - Teg Tegmentum - TG tegmental nucleus of Gudden - Tg tongue - TO tuberculum olfactorium - VT ventral thalamus - WH white matter - ZI Zona incerta - 3 third ventricle - 4 fourth ventricle - II _N Nervus opticus - Vg Trigeminal ganglion - Vsp spinal trigeminal nucleus - VIIG Ganglion geniculi - VIIIG Ganglion vestibulare - IXGI interior ganglion of IX - IXGS superior ganglion of IX - XGI inferior ganglion of X Submitted by S.E. as her doctoral thesis at the medical faculty of the University of Zürich     

Cholinergic projections to the anterior thalamic nuclei in the rat: a combined retrograde tracing and choline acetyl transferase immunohistochemical study

Retrograde transport of horseradish peroxidase (HRP) was combined with choline acetyltransferase (ChAT) immunohistochemistry to study cholinergic projections to the anterior thalamic nuclei in the rat. Small iontophoretic injections of HRP placed into different subdivisions of the anterior thalamic nuclear complex resulted in distinct patterns of retrograde labelling in two major cholinergic cell groups of the mesopontine tegmentum, the laterodorsal tegmental nucleus (LDTg), in which a majority of the labelled cells was located, and the pedunculopontine tegmental nucleus (PPT). After injections into the posterior subdivision of the anteroventral thalamic nucleus (AVp), double-labelled neurons were present predominantly in the ipsilateral LDTg while a smaller number was found in the PPT. In the ipsilateral LDTg, 60–70% of ChAT-positive neurons were HRP-labelled, and 90–95% of the HRP-labelled neurons were ChAT-positive. In the contralateral LDTg, 30–40% of ChAT-positive neurons were HRP-labelled. After injections in the medial subdivision of the anteroventral thalamic nucleus (AVm), the pattern of labelling in LDTg was similar to that detected after injections in the AVp. The number of double-labelled neurons in the LDTg and PPT was much lower after injections into AVm than after injections into AVp. When injections were confined to the anterodorsal thalamic nucleus (AD), no HRP-labelled cells were present in the LDTg or PPT. These results show that the LDTg and PPT are the sources of the cholinergic input to the rat anterior thalamus. The major projection from LDTg and PPT is to the AVp, whereas there is a lighter cholinergic projection to the AVm. The AD does not receive a projection from cholinergic cells in the mesopontine tegmentum.Abbreviations AChE Acetylcholinestrase - AD anterodorsal thalamic · nucleus - AM anteromedial thalamic nucleus - ATN anterior thalamic nuclei - AVm medial subdivision of the anteroventral thalamic nucleus - AVp posterior subdivision of the anteroventral thalamic nucleus - CG central gray - ChAT choline acetyltransferase - DAB diaminobenzidine tetrahydrochloride - DR dorsal raphé nucleus - f fornix - HRP horseradish peroxidase - ic internal capsule - LD laterodorsal thalamic nucleus - LDTg laterodorsal tegmental nucleus - ml medial lemniscus - mlf medial longitudinal fasciculus - mt mamillothalamic tract - NRS normal rabbit serum - PPT pedunculopontine tegmental nucleus - PT paratenial thalamic nucleus - Re reuniens thalamic nucleus - Rh rhomboid thalamic nucleus - RPn raphé pontis nucleus - scp superior cerebellar peduncle - sm stria medullaris thalami - st stria terminalis - TAAB glutaraldehyde - TRN thalamic reticular nucleus - VL ventrolateral thalamic nucleus - VM ventromedial thalamic nucleus - xscp decussation of superior cerebellar peduncle     

Distribution of calbindin and parvalbumin in the developing somatosensory cortex and its primordium in the rat: an immunocytochemical study

Summary Immunocytochemical techniques were used to analyze the distribution of the calcium-binding proteins calbindin and parvalbumin during the pre- and postnatal development of the rat somatosensory cortex. Calbindin occurs in most early differentiated neurons that form the primordial plexiform layer at embryonic day 14. This expression in transient; during the perinatal period, calbindin becomes immunologically undetectable within the structures derived from the primordial plexiform layer, i.e., the prospective layers I and VIb. Immunoreactive neurons are also absent from adult layers I and VIb. Calbindin is also detected in a second population of neurons which, from embryonic day 18 onwards, distributes diffusely within the cortical plate. Some neurons of this population show morphological traits of immaturity, while others show complete dendritic arborization. The definitive pattern of distribution of calbindin-immunoreactive neurons is achieved by postnatal day 22. Infragranular layers contain intensely-immunoreactive cells whose numerical density decreases during postnatal development, whereas in supragranular layers similar neurons are interspersed among numerous faintly-stained neurons.Parvalbumin is detected for the first time at postnatal day 6, within a small group of neurons located in cortical layer V, and extends afterwards through the whole thickness of the cerebral cortex. At this same postnatal stage, groups of immunoreactivepuncta are also found in layer IV of the somatosensory cortex; these puncta increase in density progressively and, at embryonic day 13, immunoreactive cells appear also grouped at this level. At this postnatal age, parvalbumin immunostaining delineates the somatosensory map in cortical layer IV. From this stage to adulthood, the number of immunoreactive neurons increases in the whole thickness of the somatosensory cortex. Barrels in layer IV become less distinct as immunoreactive cells and processes invade the septa. Layer IV in the adult somatosensory cortex appears more densely populated by parvalbumin immunoreactive neurons and puncta than in the surrounding areas.     

Colocalization of parvalbumin and calbindin D-28k in neurons including chandelier cells of the human temporal neocortex

Chandelier cells are cortical GABAergic interneurons with a unique synaptic specificity enabling them to exert a strong inhibitory influence on pyramidal cells. By using immunocytochemistry for the calcium-binding protein calbindin D-28k in the human temporal neocortex, we have found numerous immunoreactive processes that were identified as chandelier cell axon terminals. This was a striking find since in previous immunocytochemical studies of the primate neocortex, chandelier cell axon terminals had been shown to be immunoreactive for another calcium-binding protein, parvalbumin, and colocalization studies indicate that parvalbumin and calbindin are present in almost completely separate neuronal populations. Here, we present double-label immunofluorescence experiments showing that parvalbumin and calbindin immunoreactivities are colocalized in certain neurons that include a subpopulation of chandelier cells whose cell bodies are located mainly in layers V and VI of the human temporal neocortex. The results suggest a selective laminar distribution of neurochemical subtypes of chandelier cells which is a peculiar feature of the organization of the human neocortex.     

The unipolar brush cells of the rat cerebellar cortex and cochlear nucleus are calretinin-positive: a study by light and electron microscopic immunocytochemistry

Cell class-specific markers are powerful tools for the study of individual neuronal populations. The peculiar unipolar brush cells of the mammalian cerebellar cortex have only recently been definitively identified by means of the Golgi method, and we have explored markers of cerebellar neurons with the purpose of facilitating the analysis of this new cell population and, especially, its distribution and ultrastructural features. By light microscopic immunocytochemistry, we demonstrate that, in the rat, the unipolar brush cells are the cortical neurons that are most densely immunostained with antiserum to calretinin, a recently discovered calcium-binding protein. The unipolar brush cells are highly concentrated in the flocculo-nodular lobe, the ventral uvula and the ventral paraflocculus, occur at relatively high density in the lingula, at moderate-to-low density in other folia of the vermis and in the narrow intermediate cortex, and at low to very low density, with the exception of a few hot spots, in the lateral regions of the cerebellar hemispheres and in the dorsal paraflocculus. Unipolar brush cells are also found in the cochlear nucleus. In addition to the unipolar brush cells, calretinin antibody distinctly stains certain mossy fibers, and weakly to moderately stains other cerebellar elements, such as granule neurons and climbing fibers. In the lobules containing high densities of unipolar brush cells, the granule cell bodies and the parallel fibers are much less immunoreactive, and there are many more densely immunostained mossy fibers than in the lobules, where these cells are rare, which suggests some relationships between these elements. In the cerebellar nuclei, small neurons are densely immunostained, while large neurons are immunonegative.The unipolar brush cells reside nearly exclusively in the granular layer. They are small neurons, intermediate in size between granule cells and Golgi cells, and their features are remarkably similar across all lobules. They usually have a single, relatively thick dendrite of varying length that terminates in a brush-like tip consisting of several short branchlets. Utilizing a pre-embedding protocol, we have identified unipolar brush cells with the electron microscope. The cytoplasm of these cells is partially obscured by the electron dense product of calretinin immunoreaction in all regions of the soma and processes. The cells are often covered with non-synaptic appendages and contain a peculiar cytoplasmic inclusion consisting of ringlet subunits. Other characteristic components are numerous neurofilaments, mitochondria and large, dense-core vesicles. Individual brushes enter one or two glomeruli, where the dendritic branchlets establish an unusually extensive synapse with mossy fiber rosettes. In addition to their contact with the mossy rosettes, the branchlets are postsynaptic to boutons presumably belonging to the axonal plexus of Golgi cells and are also presynaptic to small dendrites, displaying small, clear synaptic vesicles at the site of contact. The distinct calretinin-like immunoreactivity of the unipolar brush cells may be related to strong calcium influx at their extensive synapses with the mossy fiber rosettes.     

Postnatal X-ray irradiation effects on glomerular layer of rat olfactory bulb: quantitative and immunocytochemical analysis

Summary In the rat olfactory bulb, the majority of interneurons in the glomerular layer (GL) are supposed to be generated during first postnatal week. Low and repeated doses of X-rays (200 rad x 4 and 200 rad x 6) were used during this period to impair the development of interneurons. The resulting effects on olfactory bulb neurons were examined stereologically and immunocytochemically in animals of 4 and 12 weeks of age. Quantitative analysis showed that, 1) the volume of the GL decreased to 55% (1200 rad) – 70% (800 rad) of control, 2) numerical cell densities in GL decreased to 40% (1200 rad) – 60% (800 rad) of control, thus resulting in 3) a decrease of the total cell number in GL to 20% (1200 rad) – 40% (800 rad) of control in irradiated olfactory bulbs of animals 4 weeks old. In comparison, mitral cells, which are generated prenatally, were much less affected (total cell number: 70–80% of control), indicating a selective loss of cells generated during the first postnatal week in GL. Effects on somata and processes immunoreactive for GABA, tyrosine hydroxylase (TH), calbindin D-28K and parvalbumin (PV) were examined in irradiated bulbs of both 4 and 12 week-old rats. All of these immunoreactive elements showed a drastic decrease in all layers. Semiquantitative analysis showed that in the GL, calbindin D-28K immunoreactive (calbindin D-28K(+)) neurons decreased more extensively than TH immunoreactive (TH(+)) and GABA-like immunoreactive (GABA(+)) neurons; that is, TH(+) and GABA(+) neurons decreased to 20% (1200 rad) – 40% (800 rad) of control, whereas calbindin D-28K(+) neurons decreased to 10% (1200 rad) – 30% (800 rad) of control in the GL of irradiated bulbs. These findings indicated that larger proportions of calbindin D-28K(+) neurons might be generated during the first postnatal week than those of GABA(+) and TH(+) neurons. Furthermore, in irradiated bulbs the proportion of GABA(-)TH(+) cells in TH(+) cells increased to about twice of control, and the estimated total numbers of GABA(-)TH(+) cells in irradiated rats were 95% (800 rad) and 40% (1200 rad) of control. These observations suggest that the majority of GABA(-)TH(+) neurons were less affected by X-ray irradiation during the first postnatal week and thus that they might be generated in the prenatal period. Since during the first 2 postnatal weeks, neurons showing GABA(-)TH(+) were not seen in GL (Kosaka et al. 1987a), the majority of GABA(-)TH(+) neurons in adult olfactory bulb were assumed to change their phenotype at some postnatal developmental period.     

GABAergic neuronal populations in monkey primary auditory cortex defined by co-localized calcium binding proteins and surface antigens

Summary The primary auditory cortex (A1) of monkeys was investigated by immunohistochemistry, using antibodies to gamma-aminobutyric acid (GABA), to the calcium binding proteins parvalbumin and calbindin, and to certain proteoglycan epitopes. The two calcium binding proteins were found to be localized in subpopulations of GABAergic neurons. Parvalbumin immunoreactive cells were mostly found in the middle layers of the cortex. Parvalbumin immunoreactivity was found in fibres in the white matter underlying A1 and a particularly dense concentration of parvalbumin immunoreactive fibers and terminals occurred in layer IV suggesting that a significant population of geniculocortical fibers is also parvalbumin positive. Calbindin positive cells were mostly located in superficial layers and in these layers the neuropil staining was also dense. Two monoclonal antibodies (MAbs) raised against monkey brain tissue and which had previously been shown to recognize neuronal surface antigens stained overlapping subpopulations of GABAergic cells. Occasional pyramidal cells were also immunoreactive. Most of the MAb positive cells were found in the middle layers and all were parvalbumin but not calbindin immunoreactive. Although the physiological roles in the brain for calcium binding proteins and the relevant cell surface markers have not yet been clarified, the presence of these markers in selected subpopulations of cells suggests the existence of functionally distinct circuits in AI cortex.     

Branching neurons in the cervical spinal cord: a retrograde fluorescent double-labeling study in the rat

Summary Branching neurons giving rise to ascending and descending collaterals were studied in the cervical spinal cord of the rat. After unilateral injection of two retrograde fluorescent tracers, i.e. DY.2HCl at T2 or more caudal levels and TB at C1 or more rostral levels, many DY-TB double-labeled neurons were found in C3 to C8. These neurons were located bilaterally throughout the spinal grey matter, as well as in the lateral spinal nucleus (LSN). However, no double-labeled neurons could be detected in the laminae I and II on either side. The double-labeled neurons must represent branching neurons giving rise to a collateral ascending to the rostral injection-site or above, and another collateral descending to the caudal injection-site or below. The descending collaterals were found to extend to various spinal levels, including the lumbosacral cord. However, most of them terminated at shorter distances from their parent cell bodies; thus 20% of the C3–C8 neurons projecting to C1 or above had a descending collateral reaching T2, 8% had a collateral reaching T9, and 3% a collateral reaching L2/L3. The ascending collaterals of the majority of the branching neurons passed into the most caudal part of the medulla oblongata, and about half of these collaterals reached the level of the rostral part of the inferior olive. In regard to the neurons located in the segments C5–C8, about 13% of those projecting to T2 or below distribute an ascending collateral restricted to C2–C4, while 29% of those had an ascending collateral to C1 or above.     

Substance P-containing hypothalamic afferents to the monkey hippocampus: an immunocytochemical,tracing, and coexistence study

In order to identify the synaptic connections of substance P-containing afferents within the hypothalamo-hippocampal projection of the monkey, we performed a combined light and electron microscopic, immunocytochemical study, made lesions of the fimbriafornix, and employed retrograde tracing using WGA-HRP. Furthermore, coexistence studies for substance P and GAD were performed to identify the putative transmitters of these hypothalamic projection neurons. A plexus of large substance P-immunoreactive terminals was identified in both the innermost portion of the molecular layer and in CA2. Axon terminals in both plexuses established exclusively asymmetric synapses with spines and dendritic shafts. Substance P-immuno-reactive boutons were degenerating 5 days after lesioning, and had disappeared 10 days after ipsilateral fimbria-fornix transection. Thus, these terminals were of extrinsic origin. In contrast, immunoreactive fibers in the outer third of the dentate molecular layer remained unaffected by the lesion. Retrograde tracing combined with immunostaining for substance P revealed the parent cell bodies of the extrinsic substance P-containing afferents in the supramammillary nucleus. Colocalization studies employing a consecutive semi-thin sections technique indicate that these large substance P-containing projection neurons lack GABA as an inhibitory transmitter. These results suggest that hypothalamic afferents of the monkey hippocampus contain substance P. Because these afferents lack GABA as an inhibitory transmitter and establish exclusively asymmetric synapses, this projection may excite hippocampal target neurons.     

An ultrastructural study of the maturation of neuronal somata in the visual cortex of the rat

Summary The postnatal development of neuronal perikarya in layers II–VI of the visual cortex of perfusion-fixed albino rats, 12 h to 180 days old, has been studied by electron microscopy. Particular attention was paid to cells in photographic montages of 75m wide strips extending through the full depth of the occipital cortex, cut from 100 m vibratome sections of the brain.At birth, and during the first few postnatal days, most of the neurons present in the cortex are small, tightly packed indifferent cells with scanty cytoplasm containing mitochondria and chiefly free ribosomes; a few presumptive pyramidal cells with a developing apical dendrite and more voluminous cytoplasm can be recognized in deep cortex. Non-pyramidal cells can be identified on postnatal day 6, when although scarce and with immature cytoplasmic features, they already display a more electron opaque chromatin pattern than developing pyramidal cells and receive axo-somatic contacts of Gray's type I.During the second postnatal week there are conspicuous increases in the maturity of the cells, which acquire a rich complement of cytoplasmic organelles: in general cells situated in the deep cortical plate are larger and better differentiated than those in the superficial plate, and non-pyramidal cells are less well differentiated than the associated pyramidal cells. By the end of the second week, differences in cytoplasmic maturity between superficial and deep, and between pyramidal and non-pyramidal cells are less evident.Maturation proceeds during the third postnatal week; both types of cells acquire an adult complement of axo-somatic synapses and their mature nuclear and cytoplasmic features, and by day 24 are indistinguishable from their adult counterparts. In keeping with previous Golgi studies of this same cortex, the non-pyramidal cells did not acquire mature ultrastructural features significantly later than the pyramidal cells. A possible correlate of particularly active synaptogenesis and plasticity in the population of nonpyramidal, cells during the third postnatal week (immediately after eyeopening), was that at this time these cells contained very prominent accumulations of granular reticulum, ribosomes and Golgi apparatus, and appeared hypertrophic.     

The relationship of calbindin-containing neurons with substance P, Leu-enkephalin and cholecystokinin fibres: An immunohistochemical study in the rat thalamus

In the rat thalamus, immunoreactivity for the calcium binding protein calbindin (Cb) is mostly confined to neuronal cell bodies, sometimes revealing proximal dendrites, of the midline, intralaminar and posterior regions. Substance P (SP)-, cholecystokinin (CCK)- and Leu-enkephalin (L-ENK)-immunoreactive (ir) elements in the thalamus are fibre-like structures, intermingled with punctate elements probably representing axonal arborizations and their synaptic boutons. These peptidergic fibres are unevenly distributed in several thalamic domains, including the areas that contain Cb-ir neurons. The relationship between Cb-ir cell bodies and these three different peptidergic systems of thalamic innervation was studied with immunohistochemistry. Single-labelling experiments on adjacent sections and double immunostaining on the same section were performed. A considerable overlap between Cb-ir perikarya and SP-ir fibres was found in most thalamic nuclei. In particular, in the intralaminar nuclei and posterior complex. SP-ir punctate elements were frequently observed in close proximity to Cb-ir cell bodies and dendrites. On the other hand, no consistent topographical correspondence between Cb-ir perikarya and CCK- or L-ENK-ir fibres was evident. Altogether, the present data suggest a selective anatomical and, possibly, functional relationship between SP and Cb in at least a subpopulation of rat thalamic neurons.     

GABA and glycine as putative transmitters in subcortical pathways to the pontine nuclei. A combined immunocytochemical and retrograde tracing study in the cat with some observations in the rat.

Using the retrograde tracers horseradish peroxidase-wheatgerm agglutinin and gold particles conjugated to wheatgerm agglutinin apo-horseradish peroxidase in combination with an antiserum against glutaraldehyde-fixed GABA, it was examined whether the pontine nuclei of the cat receive projections from GABA-like immunoreactive neurons in the brainstem, diencephalon, or deep cerebellar nuclei, contributing to the GABA-like immunoreactive fibre plexus previously demonstrated in the pontine nuclei [Brodal et al. (1988) Neuroscience 25, 27-45]. Following tracer injections that covered both the pontine nuclei and the reticular tegmental nucleus in two cats, it was found that 125 out of 1166 (10.7%) and 29 out of 294 (9.9%) retrogradely labelled neurons in the cerebellar nuclei were GABA-like immunoreactive. In the same two experiments only six out of 2029 (0.3%) and 10 out of 1398 (0.7%) retrogradely labelled neurons in the brainstem and diencephalon were GABA-like immunoreactive. Among the regions in the brainstem and diencephalon known to project to the pontine nuclei, double-labelled cells were seen in the reticular formation, the periaqueductal gray, and the nucleus praepositus hypoglossi, but not in the zona incerta or the anterior pretectal nucleus, regions that have been shown to contain glutamate decarboxylase-like immunoreactive neurons projecting to the pontine nuclei in the rat [Border et al. (1986) Brain Res. Bull. 17, 169-179]. In order to test whether this is due to species differences, the same experimental approach was used in the rat, and it was found that 54 out of 3249 (1.7%) retrogradely labelled neurons in the brainstem and diencephalon were double-labelled. Notably, in the zona incerta 2% of the retrogradely labelled cells were also GABA-like immunoreactive, and in the reticular formation there was a higher proportion of double-labelled cells than was found in the cat. Additional sources were identified, that may contribute to the GABA-like immunoreactive fibre plexus in the pontine nuclei of the rat. This, in conjunction with the previous finding that the pontine nuclei of the rat contain only very few putative GABAergic neurons [Border and Mihailoff (1985) Expl Brain Res. 59, 600-614; Brodal et al. (1988) Neuroscience 25, 27-45], lead to the suggestion that the GABA-like immunoreactive fibre plexus in the pontine nuclei of the rat is predominantly of extrinsic origin, possibly representing a mosaic of the terminal fields of several subcorticopontine projections.(ABSTRACT TRUNCATED AT 400 WORDS)     

Brain stem afferent connections of the amygdala in the rat with special references to a projection from the parabigeminal nucleus: a fluorescent retrograde tracing study

A recently revealed important function of the amygdala (Am) is that it acts as the brain’s “lighthouse”, which constantly monitors the environment for stimuli which signal a threat to the organism. The data from patients with extensive lesions of the striate cortex indicate that “unseen” fearful and fear-conditioned faces elicit increased Am responses. Thus, also extrageniculostriate pathways are involved. A multisynaptic pathway from the retina to the Am via the superior colliculus (SC) and the pulvinar was recently suggested. We here present data based on retrograde neuronal labeling following injection of the fluorescent tracer Fluoro-Gold in the rat Am that the parabigeminal nucleus (Pbg) emits a substantial, bilateral projection to the Am. This small cholinergic nucleus (Ch8 group) in the midbrain tegmentum is a subcortical relay visual center that is reciprocally connected with the SC. We suggest the existence of a second extrageniculo-striate multisynaptic connection to Am: retina–SC–Pbg–Am, that might be very effective since all tracts listed above are bilateral. In addition, we present hodological details on other brainstem afferent connections of the Am, some of which are only recently described, and some others that still remain equivocal. Following selective injections of Fluoro-Gold in the Am, retrogradely labeled neurons were observed in parasubthalamic nucleus, peripeduncular nucleus, periaqueductal gray, dopaminergic nuclear complex (substantia nigra pars lateralis and pars compacta, paranigral, parabrachial pigmented and interfascicular nuclei, rostral and caudal linear nuclei, retrorubral area), deep mesencephalic nucleus, serotoninergic structures (dorsal, median and pontine raphe nuclei), laterodorsal and pedunculopontine tegmental nuclei (Ch6 and Ch5 groups), parabrachial nuclear complex, locus coeruleus, nucleus incertus, ventrolateral pontine tegmentum (A5 group), dorsomedial medulla (nucleus of the solitary tract, A2 group), ventrolateral medulla (A1/C1 group), and pars caudalis of the spinal trigeminal nucleus. A bilateral labeling of the upper cervical spinal cord was also observed.     

Origin of projections from the midbrain raphe nuclei to the hypothalamic paraventricular nucleus in the rat: A combined retrograde and anterograde tracing study

A number of neuronal functions governed by the hypothalamic paraventricular nucleus are influenced by serotonin, and it is generally believed that the moderate density of serotonin-immunoreactive fibres and terminals within the paraventricular nucleus originates from the midbrain dorsal and median raphe nuclei. To further evaluate the intricate anatomy of projections from brain stem raphe nuclei of the rat, a combination of retrograde and anterograde tracing experiments were conducted to determine the medullary raphe nuclei projection to the paraventricular nucleus. Rhodamine-labelled latex microspheres, Cholera toxin subunit B and FluoroGold we used as retrograde tracers. Intracerebroventricular injections into the third ventricle of all retrograde tracers labelled a distinct population of neurons in the dorsal raphe situated in the subependymal stratum adjacent to the cerebral aqueduct indicating that these cells take up the tracer from the cerebrospinal fluid. Very few retrogradely labelled neurons were seen in the median raphe after i.c.v. administration of the tracers. Retrograde tracers delivered into the medial part of the paraventricular nucleus labelled no further cells in the midbrain dorsal and median raphe nuclei, whereas a substantial number of retrogradely labelled cells emerged in the pontine raphe magnus. However, when the retrograde tracers were delivered into the lateral part of the paraventricular nucleus, avoiding leakage of the tracer into the ventricle, very few labelled neurons were seen in the dorsal and median raphe, whereas the prominent labelling of raphe magnus neurons persisted. The anatomical organization of nerve fibres terminating in the area of paraventricular nucleus originating from midbrain raphe nuclei was studied in a series of anterograde tracing experiments using the plant lectin Phaseolus vulgaris leucoagglutinin. Injections delivered into the dorsal raphe or median raphe labelled but a few fibres in the paraventricular nucleus proper. A high number of fine calibered nerve fibres overlying the ependyma adjacent to the paraventricular nucleus was, however, seen after the injections into the subependymal rostral part of the dorsal raphe. Injections delivered into the raphe magnus gave rise to a dense plexus of terminating fibres in the parvicellular parts of the paraventricular nucleus and moderately innervated the posterior magnocellular part of the paraventricular nucleus as well as the magnocellular supraoptic nucleus. Concomitant visualization of serotonin-immunoreactive neurons and retrograde FluoroGold-tracing from the paraventricular nucleus revealed that none of the serotonergic neurons of the raphe magnus projects to this nucleus, while a few of the neurons putatively projecting to the paraventricular nucleus from the median raphe are serotonergic.

The current observations suggest that the raphe magnus constitute by far the largest raphe input to the paraventricular nucleus and strongly questions the earlier held view that most raphe fibres innervating the paraventricular nucleus are derived from the midbrain dorsal and median raphe. However, the source of serotonergic innervation of the paraventricular nucleus remains elusive.     

Postnatal development of parvalbumin and calbindin D28K immunoreactivities in the cerebral cortex of the rat

Parvalbumin and calbindin D28k immunoreactivities were examined in the neocortex of the rat during postnatal development. Parvalbumin-immunoreactive nonpyramidal neurons first appear in layer V and later in layers VI and IV, and then in II and III. Immunoreactive terminals forming baskets surrounding unlabelled somata appear about 2 days later. The first parvalbumin-immunoreactive neurons appear in the retrosplenial and cingulate cortices, and the rostral region of the primary somatosensory cortex at postnatal days 8 or 9 (P8–P9). These regions are followed by the primary visual, primary auditory and motor cortices at P11. Parvalbumin immunoreactivity appears last in the secondary areas of the sensory regions and association cortices. Adult patterns are reached at the end of the 3rd week. Calbindin D28K-immunoreactive nonpyramidal neurons are found at birth in all cortical layers excepting the molecular layer. The intensity of the immunoreaction increases during the first 8 or 11 days of postnatal life, first in the inner and later in the upper cortical layers, following, therefore, an inside-out gradient. Heavily-labelled calbindin D28K-immunoreactive nonpyramidal cells dramatically decrease in number from P11 to P15 due mainly to a decrease of the multipolar subtypes. This suggests that two populations of calbindin D28k-immunoreactive nonpyramidal neurons are produced in the neocortex during postnatal development: one population of neurons transitorily expresses calbindin D28k immunoreactivity; the other population is composed of neurons that are permanently calbindin D28k immunoreactive. In addition to heavily labelled nonpyramidal cells, a band of weakly labelled pyramid-like neurons progressively appears in layers II and III throughout the cerebral cortex, beginning in layer IV in the somatosensory cortex by the end of the 2st week. Adult patterns are reached at the end of the 3rd week. These results indicate that parvalbumin and calbindin D28k immunoreactivities in the cerebral neocortx follow different characteristic patterns during postnatal development. The appearance of parvalbumin immunoreactivity correlates with the appearance of the related functional activity in the different cortical regions, and, probably, with the appearance of inhibitory activity in the neocortex. On the other hand, the early appearance of calbindin D28k immunoreactivity in the neocortex may be related to the early appearance of calbindin immunoreactivity in many other brain regions, and suggests another, as yet unknown, role for this calcium-binding protein during development of the cerebral cortex.     

Paroxysmal neuronal depolarizations in the rat motor cortex in vivo: intracellular injection of the calcium agonist BAY K 8644

Summary Epileptic activity was elicited in the rat's motor cortex by local application of penicillin. At the neuronal level it consisted of typical paroxysmal depolarization shifts. The calcium agonist BAY K 8644 was injected into neurons showing such a discharge pattern. The application of this drug increased amplitude and afterdepolarization of paroxysmal neuronal depolarizations.