Perikaryal Labeling Research Articles

Immunofluorescently labeling glutamic acid decarboxylase 65 coupled with confocal imaging for identifying GABAergic somata in the rat dentate gyrus—A comparison with labeling glutamic acid decarboxylase 67

As γ-aminobutyric acid (GABA) is synthesized by two isoforms of glutamic acid decarboxylase (GAD), namely, GAD65 and GAD67, immunohistochemically targeting either isoform of GAD is theoretically useful for identifying GABAergic cell bodies. In practice, targeting GAD67 remains to be a popular choice. However, identifying GABAergic cell bodies with GAD67 immunoreactivity in the hippocampal dentate gyrus, especially in the hilus, is not without pitfalls. In the present study, we compared the characteristics of GAD65 immunoreactivity to GAD67 immunoreactivity in the rat dentate gyrus and examined perikaryal expression of GAD65 in four neurochemically prevalent subgroups of interneurons in the hilus.Experiments were done in normal adult Sprague-Dawley rats and GAD67-GFP knock-in mice. Horizontal hippocampal slices cut from the ventral portion of hippocampi were immunofluorescently stained and scanned using a confocal microscope. Immunoreactivity for both GAD67 and GAD65 was visible throughout the dentate gyrus. Perikaryal GAD67 immunoreactivity was denser but variable in terms of distribution pattern and intensity among cells whereas perikaryal GAD65 immunoreactivity displayed similar distribution pattern and staining intensity. Among different layers of the dentate gyrus, GAD67 immunoreactivity was densest in the hilus despite GAD65 immunoreactivity being more intense in the granule cell layer. Co-localization experiments showed that GAD65, but not GAD67, was expressed in all hilar calretinin (CR)-, neuronal nitric oxide synthase (nNOS)-, parvalbumin (PV)- or somatostatin (SOM)-positive somata. Labeling CR, nNOS, PV, and SOM in sections obtained from GAD67-GFP knock-in mice revealed that a large portion of SOM-positive cells had weak GFP expression. In addition, double labeling of GAD65/GABA and GAD67/GABA showed that nearly all of GABA-immunoreactive cells had perikaryal GAD65 expression whereas more than one-tenth of GABA-immunoreactive cells lacked perikaryal GAD67 immunoreactivity. Inhibition of axonal transport with colchicine dramatically improved perikaryal GAD65 immunoreactivity in GABAergic cells without significant augmentation to be seen in granule cells. Double labeling GAD65 and GAD67 in the sections obtained from colchicine-pretreated animals confirmed that a portion of GAD65-immunoreactive cells had weak or even no GAD67 immunoreactivity.We conclude that for confocal imaging, immunofluorescently labeling GAD65 for identifying GABAergic somata in the hilus of the dentate gyrus has advantages over labeling GAD67 in terms of easier recognition of perikaryal labeling and more consistent expression in GABAergic somata. Inhibition of axonal transport with colchicine further improves perikaryal GAD65 labeling, making GABAergic cells more distinguishable.

Preganglionic endings from nucleus of Edinger-Westphal in pigeon ciliary ganglion contain neuronal nitric oxide synthase.

The avian ciliary ganglion (CG) controls choroidal blood flow by its choroidal neurons, and pupil constriction and accommodation by its ciliary neurons. It was previously reported that both choroidal and ciliary neurons label positively for NADPH diaphorase (NADPHd), a marker for nitric oxide synthase (NOS). To assess if this labeling is preganglionic or postganglionic and to determine if it is attributable to neuronal NOS (nNOS), we studied pigeon CG using NADPHd histochemistry and nNOS immunohistochemistry (IHC). Short-duration staining times by NADPHd histochemistry yielded intense labeling of structures that appeared to be the cap-like endings on ciliary neurons and the boutonal endings on choroidal neurons that arise from the nucleus of Edinger-Westphal (EW), and light or no postganglionic perikaryal staining. The light postganglionic staining that was observed tended to be localized to ciliary neurons. Consistent with this, NADPHd+ nerve fibers were observed in the postganglionic ciliary nerves but rarely in the postganglionic choroidal nerves. These same staining times yielded robust staining of neurons in the orbital pterygopalatine microganglia network, which are known to be nNOS+. Diffuse staining of CG perikarya was observed with longer staining durations, and this staining tended to mask the preganglionic labeling. Preganglionic NADPHd+ staining in CG with short staining times was blocked by the NOS inhibitors iodonium diphenyl (IDP) and dichlorophenol-indophenol (DPIP), but the diffuse postganglionic staining observed with the longer staining times was not completely blocked. Labeling of CG sections for substance P (SP) by IHC (which labels EW-originating preganglionic endings in CG) and subsequently for NADPHd confirmed that NADPHd was localized to preganglionic endings on CG neurons. Immunohistochemical double labeling for nNOS and SP or enkephalin further confirmed that nNOS is found in boutonal and cap-like endings in the CG. Two studies were then carried out to demonstrate that the nNOS+ preganglionic endings in CG arise from EW. First, NADPHd+ and nNOS+ neurons were observed in EW in pigeons treated with colchicine to enhance perikaryal labeling. Second, NADPHd+ and nNOS+ preganglionic endings were eliminated from CG ipsilateral to an EW lesion. These various results indicate that NOS is present in EW-arising preganglionic endings on choroidal and ciliary neurons in avian CG. NOS also appears to be found in some ciliary neurons, but its presence in choroidal neurons is currently uncertain.

Immunohistochemical localization of adenosine A2A receptors in the rat central nervous system

The A2A adenosine receptor (A2A-AR) transcript and radioligand binding sites have a distinct distribution in rat brain, restricted primarily to the striatum, nucleus accumbens and olfactory tubercles. We describe here the use of purified recombinant human A2A-ARs to generate a monoclonal antibody that has been used to better resolve the distribution of A2A-ARs in rat brain. The antibody can detect 1 ng of purified recombinant receptor by Western blotting and is potent (EC50 = 0.62 microg/ml) and highly selective for the A2A-AR subtype. By Western blotting, the apparent molecular mass of recombinant and rat striatal receptors shifts upon deglycosylation from 43-48 to 42 kilodaltons. Analyses of chimeric A1/A2A-ARs and synthesis of a blocking peptide pinpointed the epitope (SQPLPGER) of the antibody to the center of the third intracellular loop of the receptor. Incubation of rat striatal membranes with antibody reduces receptor coupling to G-proteins. In rat brain, dense A2A-AR-like immunoreactivity that is eliminated by the blocking peptide was found in the neuropil of the striatum, nucleus accumbens (rostral pole, core and shell), cell bridges of the striatum, olfactory tubercles, and areas of extended amygdala with somewhat lighter labeling in the globus pallidus and nucleus of the solitary tract. Light perikaryal labeling was found in other areas of the brain, including the cortex, hippocampus, thalamus, cerebellum, and portions of the hindbrain. The observed distribution of A2A-AR immunoreactivity throughout the neuraxis is consistent with the receptors' role in modulating dopaminergic neurotransmission and central control of cardiovascular function.

Connections of the superior colliculus with the tegmentum and the cerebellum in the hedgehog tenrec

Different tracer substances were injected into the superior colliculus (CoS) in order to study its afferents and efferents with the meso-rhombencephalic tegmentum, the precerebellar nuclei and the cerebellum in the Madagascan hedgehog tenrec. The overall pattern of tectal connectivity in tenrec was similar to that in other mammals, as, e.g. the efferents to the contralateral paramedian reticular formation. Similarly the origin of the cerebello-tectal projection in mainly the lateral portions of the tenrec's cerebellar nuclear complex corresponded to the findings in species with little binocular overlap. In comparison to other mammals, however, the tenrec showed a consistent projection to the ipsilateral inferior olivary nucleus, in addition to the classical contralateral tecto-olivary projection. The tenrec's CoS also appeared to receive an unusually prominent monoaminergic input particularly from the substantia nigra, pars compacta. There was a reciprocal tecto-parabigeminal projection, a distinct nuclear aggregation of parabigeminal neurons, however, was difficult to identify. The dorsal lemniscal nucleus did not show perikaryal labeling in contrast to the paralemniscal region. Similar to the cat but unlike the rat there were a few neurons in the nucleus of the central acoustic tract. Unlike the cat, but similar to the rat there was a distinct, predominantly ipsilateral projection to the magnocellular reticular field known to project spinalward.

Distribution of alpha 2C-adrenergic receptor-like immunoreactivity in the rat central nervous system.

The distribution of alpha 2C-adrenergic receptors (ARs) in rat brain and spinal cord was examined immunohistochemically by using an affinity purified polyclonal antibody. The antibody was directed against a recombinant fusion protein consisting of a 70-amino-acid polypeptide portion of the third intracellular loop of the alpha 2C-AR fused to glutathione-S-transferase. Selectivity and subtype specificity of the antibody were demonstrated by immunoprecipitation of [125I]-photoaffinity-labeled alpha 2-AR and by immunohistochemical labeling of COS cells expressing the individual rat alpha 2-AR subtypes. In both cases the antibody recognized only the alpha 2C-AR subtype, and immunoreactivity was eliminated by preadsorption of the antibody with excess antigen. In rat brain, alpha 2C-AR-like immunoreactivity (alpha 2C-AR-LI) was found primarily in neuronal perikarya, with some labeling of proximal dendrites; analysis by confocal microscopy revealed the intracellular localization of some of the immunoreactivity. Areas of dense immunoreactivity include anterior olfactory nucleus, piriform cortex, septum, diagonal band, pallidum, preoptic areas, supraoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, amygdala, hippocampus (CA1 and dentate gyrus), substantia nigra, ventral tegmental area, raphe (pontine and medullary), motor trigeminal nucleus, facial nucleus, vestibular nucleus, dorsal motor nucleus of the vagus, and hypoglossal nucleus. Labeling was found in specific laminae throughout the cortex, and a sparse distribution of very darkly labeled cells was observed in the striatum. At all levels of the spinal cord there were small numbers of large, darkly labeled cells in layer IX and much smaller cells in layer X. In general, the pattern of alpha 2C-LI throughout the neuraxis is consistent with previously published reports of the distribution of receptor mRNA detected by hybridization histochemistry.

Abnormal perikaryal accumulation of neurofilament light protein in the brain of mice transgenic for the human protein: sequence of postnatal development.

Adult mice transgenic for the human form of neurofilament light protein display abnormal perikaryal immunoreactivity for this protein in many regions of the CNS and notably the thalamus. To determine the sequence of development of these anomalies, we have compared normal and transgenic mice of different postnatal ages (P0-P70), using immunocytochemistry with primary antibodies recognizing both murine and human sequence of neurofilament light protein (NR-4) or the human form only (DP5-1-12). In normal mouse brainstem, several nuclei displayed immunoreactive perikarya at P0. The number of these perikarya culminated at P10, followed by a general decrease, some nuclei having lost all perikaryal immunostaining in adults. In transgenic mouse brainstem, the distribution of perikaryal immunoreactivity already resembled at P0 that of P10 in normal mouse, and remained unchanged in adults. Differences between normal and transgenic mice were even more pronounced in the forebrain. Some nuclei of normal mouse basal forebrain that were weakly immunopositive at P10 or P20, but no longer in adults, were already labeled at P0 and remained so or became more intense at later stages in transgenic mice. In the thalamus of normal mouse, perikaryal labeling was faint, confined to a few nuclei, and detected only transiently at P10, whereas in transgenics, it was already observed in some nuclei at P0, increased in intensity and extended to other nuclei at P10, and persisted thereafter. Strongly immunoreactive, inflated perikarya with excentric nuclei were prominent in these thalamic nuclei at P20, and even larger in size at P70. In the cerebral cortex of normal mice, layers II-III and layer V of many cytoarchitectonic areas showed immunoreactive cell bodies at P10, a distribution which became gradually restricted to the parietal cortex in adults. In transgenic mice, immunopositive cortical cell bodies were first detected at P3, filled layers II-III of numerous cortical areas at P10, and then rapidly decreased in number to approach the adult pattern at P20. In the cortex as well as thalamus of P10 transgenic mice, differences between the patterns of cellular staining with clones NR4 and DP5-1-12 antibodies indicated that both the murine and human proteins were accumulated in these neurons. Thus, neurofilament light protein accumulation in the transgenic mouse brain generally involved neurons displaying perikaryal immunoreactivity for the protein at least at some point during normal postnatal development.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunocytochemical localization of the renal neutral and basic amino acid transporter in rat adrenal gland, brainstem, and spinal cord

A neutral and basic amino acid transporter (NBAT) cloned from rat kidney was recently localized to enteroendocrine cells and enteric neurons. We used an antibody directed against a synthetic peptide representing a putative extracellular domain of NBAT to determine whether this transporter was also present in other endocrine and neural tissues, including rat adrenal gland, brainstem, and spinal cord. Abundant, highly granular labeling for NBAT was observed in the cytoplasm of chromaffin and ganglion cells in the adrenal medulla. A small population of intensely labeled varicose processes was also seen in both the cortex and the medulla of the adrenal gland. More numerous, intensely labeled varicose processes were detected in brainstem and spinal cord nuclei, including the locus coeruleus, rostral ventrolateral medulla, nuclei of the solitary tract, dorsal motor nucleus of the vagus, and intermediolateral cell column of the thoracic spinal cord. Significant perikaryal labeling for NBAT was only detected in brainstem and spinal cord following intraventricular colchicine treatment, which increased the number, distribution, and intensity of NBAT-immunolabeled cells. These NBAT-immunoreactive perikarya were most numerous in the locus coeruleus, rostral ventrolateral medulla, nuclei of the solitary tract, and raphe nuclei. Ultrastructural examination of the nuclei of the solitary tract of normal rats showed that NBAT was localized predominantly to axon terminals. Within these labeled terminals, NBAT was associated with large dense core vesicles and discrete segments of plasma membrane. The observed localization of NBAT suggests that this renal specific amino acid transporter subserves a role as a vesicular or plasmalemmal transporter in monoamine-containing cells, including chromaffin cells and autonomic neurons.

Patterns of inter- and intralaminar GABAergic connections distinguish striate (V1) and extrastriate (V2, V4) visual cortices and their functionally specialized subdivisions in the rhesus monkey

Local GABAergic connections are undoubtedly important for the operation of cerebral cortex, including the tuning of receptive field properties of visual cortical neurons. In order to begin to correlate specific configurations of GABAergic networks with particular receptive field properties, we examined the arrangement of GABAergic neurons projecting to foci in compartments of known functional specialization in striate (area V1) and extrastriate (areas V2, V4) cortices of rhesus monkeys. GABAergic cells were detected autoradiographically following microinjections into supragranular, granular, or infragranular layers of 5, 10, or 50 nl of 3H-nipecotic acid, which selectively exploits the GABA reuptake mechanism. These injections produced complex inter- and intralaminar distributions of retrograde perikaryal labeling that was selective for GABA-immunopositive neurons and glia. The pattern of retrograde labeling depended on both the laminar and cytoarchitectonic location of injection sites. In all cases, a high density of labeled neurons was present in the immediate vicinity of injection sites, with the density of labeled neurons decreasing for the most part uniformly with horizontal distance. Injections in supragranular layers produced relatively widespread labeling (up to 1.5-1.7 mm from the center of injections) in upper layers, whereas in granular and infragranular layers, labeling was confined to a radius of 0.25-0.5 mm. Conversely, injections in infragranular layers produced labeling that was widest (up to 1 mm) in lower layers, but more laterally restricted in supragranular layers. Injections in granular layers, on the other hand, produced an even distribution of labeling, 0.6-1.0 mm in diameter, throughout all layers. Comparably placed injections in V1, V2, and V4 resulted in patterns of labeling that were distinguished by features including stepwise increases in the lateral extent of labeling from striate to extrastriate areas, and the circular versus markedly elongated intralaminar distribution of labeled neurons in V1 and V4 versus V2. Further, for superficial injections, labeling was present in all layers in V1 and V2, but did not extent below the top layer V in area V4. These findings offer clear examples of organizational differences in the intrinsic inhibitory connections of visual cortices. The results also demonstrate that the number of GABAergic neurons projecting to any spot in cortex decreases systematically with horizontal distance from the spot, and that radiolabeled cells do not coalesce to form slabs, columns, or clusters. This relatively even distribution of retrogradely labeled cells in the tangential plane is consistent with recent computer simulations (Worgotter and Koch, 1991) that suggest that inhibitory neurons broadly tuned as a population can produce the specific response properties of cortical neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Transient expression of somatostatin peptide is a widespread feature of developing sensory and sympathetic neurons in the embryonic rat.

Previous studies from this and other laboratories demonstrated that many embryonic sensory ganglion cells in the rat transiently express the catecholamine synthesizing enzyme tyrosine hydroxylase (TH), a trait not expressed by most mature sensory neurons. We, therefore, sought to determine whether transient expression was uniquely associated with catecholaminergic traits, or, alternatively, whether embryonic ganglion cells transiently expressed peptidergic properties as well. Of the four peptides examined (somatostatin [somatotropin release inhibiting factor] (SRIF), galanin (Gal), calcitonin gene-related peptide (CGRP), and substance P (SP)), only SRIF was found to be transiently expressed during early stages of sensory gangliogenesis. Surprisingly, SRIF immunoreactivity was observed in virtually all cranial and spinal sensory ganglion cells on embryonic day (E) 12.5. In addition to perikaryal labeling, intense SRIF immunoreactivity was also observed in the central and peripheral processes of E12.5 sensory neurons, suggesting the peptide may be released from nerve endings. The time course of SRIF appearance in cranial ganglion cells paralleled that previously described for TH, and double-labeling studies revealed extensive co-localization of these two phenotypes. By E16.5, however, the number of neurons expressing SRIF had diminished markedly, indicating that SRIF is only transiently expressed by most sensory neurons during early stages of ganglion development. An unexpected finding was that transient expression of SRIF is also a prominent feature of sympathetic ganglion cells; however, the temporal pattern of staining in the sympathetic and sensory ganglia differed substantially. Whereas virtually no SRIF staining was observed in E12.5 sympathetics, the vast majority of cells in the E16.5 superior cervical ganglion (SCG) were labeled. This contrasted sharply with the adult SCG, in which only low levels of SRIF expression were found. These findings demonstrate that SRIF peptide is transiently expressed at high levels in peripheral sensory and sympathetic neurons during embryogenesis. The time course and widespread distribution of SRIF expression indicates that the peptide may play a role in early stages of ganglion cell growth and development. Moreover, these data, in conjunction with previous studies demonstrating SRIF immunoreactivity in developing central neurons, suggest that transient expression of this peptide is a common property of diverse neuronal cell types.

Interlaminar and lateral excitatory amino acid connections in the striate cortex of monkey

The intrinsic excitatory amino acid pathways within the striate cortex of monkeys were studied by autoradiographic detection of retrogradely labeled somata following microinjections of D-3H-aspartate (D-3H-Asp) into different layers. The labeled amino acid was selectively accumulated by subpopulations of neurons and, to a small extent, by glial cells, the latter mainly in the supragranular layers. Immunocytochemical detection of neurons containing GABA showed that, apart from a few cells exclusively in layer I, GABAergic neurons do not accumulate D-3H-Asp. Several lines of evidence suggest that D-3H-Asp uptake occurred only at nerve terminals; thus, the pattern of perikaryal labeling allowed the delineation of interlaminar and lateral projections. Neurons in layer I probably project laterally, and layer I receives wide-ranging projections from layer IVB and layer V from cells up to 1300 microns laterally. Some neurons in layer II send a focused projection to lower layer VI. Some neurons in layers II/III project up to 1 mm laterally within their own layer, but relatively few neurons can be labeled in these projections. Similarly, in layers II/III few neurons can be retrogradely labeled from layers V and upper VI, and this projection is organized such that cells closer to the pia project deeper in layer V/VI. The connections of layer IVA could not be revealed separately because of the difficulty of confining injections to this thin sublamina. Neurons in layer IVB project up to 1300 microns within IVB itself. A small number of cells from IVB also project to layers III, IVC-alpha, V, and VI with much more restricted lateral spread. Neurons in upper IVC-alpha send axons to layer IVB with at least 600-800 microns lateral spread. Neurons in lower IVC-alpha/upper IVC-beta project to layer III with at least 300-500 microns lateral spread. The bottom 50-80 microns of layer IVC-beta contains neurons with a very focused projection, apparently exclusively to the layer III/IVA border region. Both layers IVC alpha and beta have rich connections within themselves, the beta sublayer having more restricted lateral connections. Some neurons in layer IVC-beta give a laterally restricted small input to layers IVC-alpha and IVB. Both IVC-alpha and -beta project to layers V and VI, and these projections are spread at least 400 microns laterally. Neurons in layer V project to all layers, but the projection to layers I-III and within layer V itself spread much further laterally than the projections to layers IV and VI.(ABSTRACT TRUNCATED AT 400 WORDS)

Afferents to the rat red nucleus studied by means of d-[ 3H] aspart ate, [ 3H]choline and non-selective tracers

Following injection of horseradish peroxidase-labeled wheat germ agglutinin or of rhodaminelabeled microspheres as non-selective tracers into the rat red nucleus, the origins of the corticorubral and cerebellorubral pathways, as well as a considerable number of other brain structures including dorsal raphénucleus, zona incerta and several hypothalamic nuclei showed retrogradely labeled perikarya. Labeling patterns obtained with horseradish peroxidase-labeled wheat germ agglutinin compared well with those observed following application of rhodamine-labeled microspheres which produced injection sites restricted to the small nucleus. In these latter cases, counterstaining with phosphine allowed a better definition of anatomical structures. After d-[ 3H]aspartate application, retrogradely labeled perikarya were observed in cerebral cortex (layer V), zona incerta, dorsal raphénucleus and in several other structures also labeled by non-selective tracers. Following application of [ 3H]choline and using an improved autoradiographic method, perikaryal labeling was massive within nucleus interpositus, while it was absent in dorsal raphénucleus, cerebral cortex and zona incerta. Retrograde tracing experiments with d-[ 3H]aspartate and [ 3H]choline revealed that these transmitter related compounds are selective markers for two subsets of afférents to the red nucleus. The transmitter specificity of the selective labeling with [ 3H]choline in the cerebellorubral pathway is supported only in part by the results obtained with other methods. The selective labeling with d-[ 3H]aspartate in the corticorubral pathway, on the other hand, is consistent with its transmitter specificity.

Immunohistochemical study of glutaminase‐containing neurons in the cerebral cortex and thalamus of the rat

In an attempt to identify glutamatergic neurons, the cerebral cortex and thalamus of the rat were examined immunohistochemically by using a monoclonal antibody against phosphate-activated glutaminase (PAG), a major synthetic enzyme of transmitter glutamate in the central nervous system. In both the neocortex and mesocortex, pyramidal cells in layers V and VI showed intense PAG-like immunoreactivity (PAG-LI), whereas neuronal cell bodies in layers I-IV showed weak PAG-LI. At the deep border of layer VI, neurons with horizontally elongated cell bodies showed PAG-LI. In the pyriform and entorhinal cortices, neurons with intense to moderate PAG-LI were seen in layer II as well as in the deeper layers. In the hippocampal formation, pyramidal cells in CA1, CA2, and CA3 and polymorphic cells in CA4 showed PAG-LI; PAG-LI was most intense in pyramidal cells of CA3. Fine granules with weak PAG-LI were also seen on and/or within the cell bodies of granule cells in the dentate gyrus. In the thalamus, neurons with PAG-LI were distributed in all nuclei, although regional differences were observed in the distribution pattern of neurons with PAG-LI and in the intensity of PAG-LI in individual neurons. The largest neurons in each thalamic nucleus showed intense PAG-LI; these were considered to be projection neurons. In addition to perikaryal labeling, many fine, PAG-like immunoreactive granules were distributed in the neuropil of both the cerebral cortex and thalamic nuclei. Some of these fine granules with PAG-LI in the neuropil were assumed to represent fiber terminals with PAG-LI, because the distribution pattern of the deposits in the primary somatosensory and primary visual cortices resembled that of thalamocortical fiber terminals. Glutamate is rather ubiquitous in the mammalian central nervous system, and it is still debatable whether the monoclonal antibody to PAG from brain mitochondria can distinguish transmitter-related glutaminase from the other metabolism-related ones. In the present study, however, large neurons in the thalamic nuclei, as well as pyramidal neurons in the cerebral cortex, showed PAG-LI most intensely, supporting the assumption that projection neurons of the cerebral cortex and thalamus are primarily glutamatergic.

Glycine: an alternative transmitter candidate of the pallidosubthalamic projection neurons in the rat.

Autoradiographic retrograde tracing techniques with radioactive transmitters were used to analyse the identity of a putative transmitter in the rat pallidosubthalamic (GP-STN) pathway. One to 2 hours after the stereotaxic injection of 3H-glycine restricted to the STN, a large number of neuronal somata were radiolabeled in the GP. No comparable labeling was observed following the injection of 3H-gamma-aminobutyric acid (3H-GABA) into the same nucleus even with survival times as long as 6 hours. Specifically, no significant somatic labeling was detected either in the GP or in the caudoputamen (CPU). Only when 3H-GABA was injected into the substantia nigra did CPU and GP neurons become labeled. On the contrary, STN neuronal somata were invariably labeled 6 hours after the intrapallidal injection of 3H-GABA, whereas no perikaryal labeling was observed in the STN after 3H-glycine injection into the GP. The perikaryal labeling was prevented in all cases by intraventricular administration of colchicine 1 day before the isotope injections. The observations suggest that 3H-glycine was preferentially transported retrogradely through the GP-STN pathway, and 3H-GABA through the STN-GP projection. In view of the recent controversy on the role of GABA as a putative transmitter of the GP-STN projection, we now propose glycine as an alternative transmitter candidate of these critically situated neurons in the basal ganglia.

Vacuolated anterior horn cells in wobbler mouse motor neuron disease: peripheral axons and regenerative capacity.

We investigated whether vacuolated cervical anterior horn cells of the wobbler mouse maintain axons to the periphery, and if these morphologically abnormal neurons are capable of supporting axonal regeneration. Using retrograde axonal transport, we applied horseradish peroxidase (HRP) to peripheral nerves or muscles and with electron microscopy sought evidence for perikaryal labeling in vacuolated neurons in 23 wobbler mice. When HRP was injected into forelimb muscles, 12 of 36 vacuolated neurons became positively labeled indicating that these neurons have axons in continuity with the periphery. In regeneration studies, after nerve crush at the brachial plexus, 23 out of 85 vacuolated neurons were labeled after HRP application at the elbow level. However, after a sufficient regeneration period, none of the 36 vacuolated neurons were labeled if HRP was applied in muscles below the elbow. In all experiments, morphologically normal neurons were always labeled. Our studies indicate that some vacuolated neurons of wobbler mice not only maintain axons into the periphery, but are also capable of supporting regeneration. However, the overall function of these vacuolated neurons appears marginal compared with the majority of morphologically normal neurons in this motor neuron disease.

Immunoreactive dynorphin B in sacral primary afferent fibers of the cat

Immunocytochemical analysis of the distribution of dynorphin B terminals in the sacral spinal cord of the cat revealed a pattern of staining very similar to that produced with antisera directed against the primary afferent derived, putative neurotransmitter, vasoactive intestinal polypeptide. Labeled axons and terminals were concentrated in lamina I and V and there was dense fiber staining in the tract of Lissauer. Of particular interest was the presence of immunoreactive axons in attached dorsal rootlets. To specifically focus on the possibility that some of the sacral primary afferent fibers are dynorphin-immunoreactive, we first tried to increase perikaryal labeling in the sacral dorsal root ganglia by topical treatment with colchicine. This did not produce immunoreactive labeling of cell bodies in the ganglia. Unilateral multiple dorsal rhizotomy (L5 to coccygeal 1), however, significantly decreased the staining of dynorphin-immunoreactive axons and terminals in the tract of Lissauer and in the dorsal horn of sacral segments ipsilateral to the deafferentation. No changes were detected in the lumbar cord. Finally, radioimmunoassay of caudal lumbar and sacral dorsal root ganglia was performed. Measurable immunoreactivity was found in all ganglia assayed, but, consistent with the histochemical analysis, sacral ganglia contained the highest concentration of immunoreactive dynorphin B. These data indicate that a significant component of the sacral spinal cord dynorphin terminal immunoreactivity derives from primary afferent fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective retrograde labeling with D‐[3H]‐aspartate in afferents to the mammalian superior colliculus

The selectivity previously reported for retrograde labeling patterns obtained following D-[3H]-aspartate injections and proposed to be related to the transmitter specificity of the labeled pathways was tested in afferents to the superior colliculus (SC) of rats and rabbits. In rats selectivity was assessed by comparing retrograde perikaryal labeling patterns observed in D-[3H]-aspartate experiments with those found after administration of a nonselective tracer, horseradish-peroxidase-labeled wheat germ agglutinin (HRP-WGA), and of the tritiated neurotrasmitter gamma-aminobutyric acid (GABA). Following D-[3H]-aspartate injections into the SC labeling was intense in a large number of cortical and hypothalamic neurons. Other afferents to the SC, however, such as those originating from the ventrolateral geniculate nucleus, the pars reticulata of the substantia nigra, the locus coeruleus, the pontine nuclei, or the retinal ganglion cells, were not labeled. Similar results were obtained in rabbits. In cats, the analysis was focused on the cerebral cortex, since in an earlier investigation no retrograde labeling had been detected in the visual cortex following D-[3H]-aspartate injections in the SC. In the present work, however, retrogradely labeled neurons were observed in various cortical areas including a few in visual cortex. This report shows selective retrograde labeling for a part of the afferents to the SC. This selectivity does not display major differences among the mammalian species studied. Moreover, according to the information available about the distribution of neurotransmitters in the brain, the findings reported here favour the idea that D-[3H]-aspartate is a retrograde tracer selective for glutamatergic and/or aspartatergic pathways.

‘Homing’ of Lucifer Yellow liposomes into hypothalamic neurons: a combined neuroanatomical Golgi and tracing technique

Liposomes are combined with Lucifer Yellow in order to evoke neuronal uptake of this fluorescent dye in the medial basal hypothalamus. Lucifer Yellow liposomes are preferentially taken up by hypothalamic neurons. The dye spreads into the dendrites and axons, producing Golgi-revealing views of several neurons near the injection site in which both untrapped and liposome-entrapped Lucifer Yellow has been microiontophoretically administered. The capriciousness of the labeled neurons makes it possible to study the dendritic arborization and spines, while the axon with its collaterals can be followed to their targets. Moreover, the dye is also taken up retrogradely. When retrograde uptake over great distances occurs, the Golgi-like appearance is missing, because only then is perikaryal labeling found.

Organization and efferent projections of nucleus tegmenti pedunculopontinus pars compacta with special reference to its cholinergic aspects

In an attempt to evaluate the cellular organization and efferent projections of the nucleus tegmenti pedunculopontinus pars compacta, several experiments were performed in the rat. From measurements of neurons in the nucleus tegmenti pedunculopontinus pars compacta in Nissl-stained sections, the nucleus was observed to contain many large neurons which made it possible to demarcate this nucleus from surrounding pontomesencephalic reticular formation. Two other neuronal populations, medium and small neurons, were also seen in the nucleus tegmenti pedunculopontinus pars compacta. Detailed measurements showed that 90% by volume of all neurons in the nucleus tegmenti pedunculopontinus pars compacta were large and medium-sized neurons. After injections of [ 3H]leucine into the nucleus tegmenti pedunculopontinus pars compacta, transported label was observed in dorsally and ventrally coursing ascending fibers. The dorsally coursing fibers entered the centrolateral nucleus and centre median-parafascicular complex of the thalamus. The ventrally coursing fibers produced accumulation of silver grains in the ventral tegmental area, substantia nigra pars compacta, subthalamic nucleus, zona incerta and lateral hypothalamus. Crossed fibers of the nucleus tegmenti pedunculopontinus pars compacta were observed sparsely at the levels of the thalamus and posterior commissure, and to a greater degree through the supraoptic commissure of Meynert. Much less anterograde labeling was seen in the equivalent terminal sites on the contralateral side of the brain. By electron microscopic autoradiography major terminal sites of axons of the nucleus tegmenti pedunculopontinus pars compacta were examined in rats injected with [ 3H]leucine in the nucleus tegmenti pedunculopontinus pars compacta and later injected with horseradish peroxidase in the striatum and pallidum. Statistical data showed preferential radiolabeling of terminals forming asymmetrical synaptic contact with dendrites in the centrolateral nucleus, centre median-parafascicular complex and subthalamic nucleus. Apparent terminations in the substantia nigra pars compacta proposed in earlier studies and shown in the present light microscopic autoradiograms were not supported by this ultrastructural analysis. Several radiolabeled terminals of the asymmetrical type contacting horseradish peroxidase labeled dendrites in the thalamus confirmed direct input from the nucleus tegmenti pedunculopontinus pars compacta to the thalamostriate projection neurons. [ 3H]choline injections into the thalamus and subthalamic nucleus produced retrograde perikaryal labeling of large neurons in the nucleus tegmenti pedunculopontinus pars compacta. These neurons were unlabeled after [ 3H]choline injections in the substantia nigra. Other findings suggested retrograde transport of [ 3H]choline through cholinergic terminals as well as cholinergic fibers of passage. These data suggested a selective uptake mechanism for cholinergic fibers of passage. The results emphasize the cholinergic nature of the nucleus tegmenti pedunculopontinus pars compacta innervation of the thalamus and subthalamic nucleus. Large neurons in the nucleus tegmenti pedunculopontinus pars compacta seem responsible for this cholinergic innervation and probably provide the axon terminals making asymmetrical synapses in the thalamus and subthalamic nucleus as described above. In addition, large neurons as well as medium and small ones in the nucleus tegmenti pedunculopontinus pars compacta whose transmitters and exact destinations remain unknown send a number of axons through the supraoptic commissure of Meynert to innervate the contralateral subthalamic nucleus.

Retrograde transport of gamma-amino[3H]butyric acid reveals specific interlaminar connections in the striate cortex of monkey.

Several lines of evidence suggest that gamma-aminobutyric acid is an inhibitory neurotransmitter in the cerebral cortex. To study the intracortical projection of neurons that selectively accumulate this amino acid, we injected radioactive gamma-aminobutyric acid into the upper layers of the striate cortex of monkeys along tracks at an oblique angle to the pia. Sections from the injected area were then processed by a combination of autoradiography and Golgi impregnation to reveal the distribution of labeled neurons and their morphological characteristics. Labeled neurons always occurred around the injection site in each layer. In addition, a consistent radial pattern of perikaryal labeling was observed in layers IVc-VI below the injection track in layers I-IVa. The closer the injection track was to the pia the deeper the peak density of labeled cells appeared. After injection in layers IVa and the lower part of III, the highest number of labeled neurons was in layer IVc; after injection in the upper part of layer III, most labeled neurons were in layer V; and, after injection in layers I and II, the proportion of labeled neurons increased in the lower part of layer V and in layer VI. All these neurons in the infragranular layers are presumably labeled by retrograde axonal transport via the labeled fiber bundles that extended from upper to lower layers. Thirty-four Golgi-stained neurons of various types were also examined for retrograde labeling. Two were labeled, and both were aspiny stellate cells in layer V. The arrangement of these putative GABAergic neurones, with axons that ascend from lower to upper layers in a regular pattern and arborize locally, would enable them to mediate inhibition within cortical columns and between neighboring columns.

3H]-GABA selective retrograde labeling of neurons in the pigeon thalamo-Wulst pathway.

Evidence is presented which is consistent with a specific retrograde labeling of GABAergic neurons following [3H]-GABA application in their zone of termination. [3H]-GABA injection in the pigeon Wulst leads to perikaryal retrograde labeling in the ipsilateral thalamic visual relay, n. dorsolateralis anterior thalami, pars lateralis (DLLv). This result gives further support to the biochemical evidence of the existence, in the pigeon, of a GABAergic projection from DLLv to the ipsilateral visual Wulst.