Charles R. Gerfen

An anterograde neuroanatomical tracing method that shows the detailed morphology of neurons, their axons and terminals: Immunohistochemical localization of an axonally transported plant lectin, Phaseolus vulgaris-leucoagglutinin (PHA-L).

A new neuroanatomical method for tracing connections in the central nervous system based on the anterograde axonal transport of the kidney bean lectin, Phaseolus vulgaris-leucoagglutinin (PHA-L) is described. The method, for which a detailed protocol is presented, offers several advantages over present techniques. First, when the lectin is delivered iontophoretically, PHA-L injection sites as small as 50-200μm in diameter can be produced, and are clearly demarcated since the neurons within the labeled zone are completely filled. Second, many morphological features of such filled neurons are clearly demonstrated including their cell bodies, axons, dendritic arbors and even dendritic spines. Third, there is some evidence to suggest that only the neurons at the injection site that are filled transport demonstrable amounts of the tracer, raising the possibility that the effective injection site can be defined quite precisely. Fourth, even with the most restricted injections, the morphology of the labeled axons and axon terminals is clearly demonstrated; this includes boutons en passant, fine collateral branches, and various terminal specialization, all of which can be visualized as well as in the best rapid Golgi preparations. Fifth, when introduced iontophoretically, PHA-L appears to be transported preferentially in the anterograde direction; only rarely is it transported retrogradely. Sixth, PHA-L does not appear to be taken up and transported effectively by fibers of passage. Seventh, there is no discernible degradation of the transported PHA-L with survival times of up to 17 days. Finally, since the transported marker can be demonstrated with either peroxidase or fluorescent antibody techniques, it may be used in conjunction with other neuroanatomical methods. For example, double anterograde labeling experiments can be done using the autoradiographic method along with immunoperoxidase localization of PHA-L, and the retrogradely transported fluorescent dyes can be visualized in the same tissue sections as PHA-L localized with immunofluorescence techniques.

Distribution of striatonigral and striatopallidal peptidergic neurons in both patch and matrix compartments: an in situ hybridization histochemistry and fluorescent retrograde tracing study

In situ hybridization histochemistry using cDNA oligonucleotide probes for the neuropeptides dynorphin, enkephalin and substance P was used to map the distribution of peptidergic neurons in the striatal patch and matrix compartments in the rat. Striatal neurons containing message for each of these peptides were distributed in both striatal compartments in the following proportions: dynorphin, in 52% of patch neurons and 45% of matrix neurons; enkephalin, in 65% of patch neurons and 58% of matrix neurons and substance P, in 61% of patch neurons of 54% matrix neurons. Fluorescent retrograde axonal tracing combined with in situ hybridization histochemistry demonstrated that the majority of neurons expressing enkephalin project to the globus pallidus and few project to the substantia nigra, whereas the reverse obtains for neurons expressing dynorphin and substance P.

Levodopa replacement therapy alters enzyme activities in striatum and neuropeptide content in striatal output regions of 6-hydroxydopamine lesioned rats

The effects of striatal dopamine denervation and levodopa replacement therapy on neuronal populations in the rat striatum were assessed by measurement of glutamic acid decarboxylase (GAD) and choline acetyltransferase (CAT) activities in the striatum, dynorphin and substance P concentrations in the substantia nigra, and enkephalin concentration in the globus pallidus. Rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal pathway were treated for 21 days with levodopa (100 mg/kg/day, i.p., with 25 mg/kg benserazide) on either an intermittent (b.i.d.) or continuous (osmotic pump infusion) regimen and sacrificed following a three day drug washout. In saline-treated control rats, striatal GAD activity and globus pallidus enkephalin content were elevated and nigral substance P content was reduced ipsilateral to the 6-OHDA lesion. Intermittent levodopa treatment further increased GAD activity, decreased CAT activity, restored substance P to control levels, markedly increased dynorphin content, and had no effect on enkephalin. In contrast, continuous levodopa elevated globus pallidus enkephalin beyond the levels occurring with denervation, but had no effect on any of the other neurochemical measures. These results indicate that striatal neuronal populations are differentially affected by chronic levodopa therapy and by the continuous or intermittent nature of the treatment regimen. With the exception of substance P, levodopa did not reverse the effects of the 6-OHDA lesion but, rather, either exacerbated the lesion-induced changes (e.g. GAD and enkephalin) or altered neurochemical markers which had been unaffected by the lesion (e.g. CAT and dynorphin).(ABSTRACT TRUNCATED AT 250 WORDS)

Substance P (neurokinin-1) receptor mRNA is selectively expressed in cholinergic neurons in the striatum and basal forebrain.

In the striatum substance P (neurokinin-1) receptor, mRNA is selectively localized in large neurons that also express mRNA encoding choline acetyltransferase (ChAT) by in situ hybridization histochemistry. Substance P receptor mRNA is also localized in ChAT mRNA-containing neurons in the medial septum and basal forebrain cell groups. Thus, in the rat forebrain the substance P receptor appears to be expressed selectively by cholinergic neurons. Striatal neurons that contain substance P also utilize gamma-aminobutyric acid (GABA) as a transmitter. These neurons make synaptic contact with striatal cholinergic neurons, which are shown here to express the substance P receptor, and with other GABAergic neurons in the striatum and substantia nigra, which express GABA receptors but not substance P receptors. This suggests that individual striatal neurons may differentially affect target neurons dependent on the receptors expressed by those target neurons.

Visceral cortex: A direct connection from prefrontal cortex to the solitary nucleus in rat

Abstract Injections of the fluorescent retrograde axonal tracer, True Blue, into the solitary complex (a visceral and taste center in the medulla) labeled pyramidal cell bodies deep in the medial, prefrontal cortex and especially lateral prefrontal (or insular) cortex of the rat. Injections of the anterograde axonal tracer (wheat germ agglutinin conjugated to horseradish peroxidase) into the prefrontal cortex produced bilateral labeling throughout much of the solitary nucleus. The distribution of prefrontal cortical cells projecting directly to the solitary nucleus may provide one clear anatomical criterion for identifying regions of cerebral cortex that may influence visceral function.

A note on the transneuronal transport of wheat germ agglutinin-conjugated horseradish peroxidase in the avian and rodent visual systems

SummaryWhile using horseradish peroxidase conjugated to the plant lectin wheat germ agglutinin (WGA-HRP) as an anterograde marker to label the developing retinofugal projection in the chick, we have found that a significant amount of the tracer can escape from the axons and axon terminals of retinal ganglion cells and be subsequently taken up and transported both anterogradely and retrogradely by neighboring neurons and/or axon terminals. The release and uptake of the tracer appears to be nonspecific, and is particularly striking at embryonic stages; at these stages there is also appreciable uptake of the WGA-HRP by ependymal cells and by radial glial processes. Subsequent experiments in rats have shown that as early as 2 days after an injection of WGA-HRP into an eye there is clear labeling of geniculo-cortical axons in the striate cortex. Since WGA-HRP is being used increasingly in neuroanatomical studies, it is now clear that when interpreting the results of experiments with this marker the possibility of transneuronal labeling must be borne in mind, and especially after relatively long post-labeling survival periods. At the same time the secondary transport of WGA-HRP may, in certain neural systems, provide a useful tool for analyzing second-order connections.

The development and restriction of the ipsilateral retinofugal projection in the chick

Although it is generally believed that the central projections of the retina in birds are entirely crossed, using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) as an anterograde tracer, we have found that in normal posthatched chicks there is a small ipsilateral retinofugal projection to the diencephalon and midbrain. Most of the ipsilateral fibers appear to be directed to the lateral anterior and dorsolateral anterior nuclei of the thalamus, to the pretectal region, and to the ectomammillary nucleus and the adjoining nucleus externus. Even in the best preparations the numbers of ipsilateral fibers are so small that it is hardly surprising that they have been overlooked in previous axonal degeneration and autoradiographic experiments. A significantly larger ipsilateral retinal projection develops during the second week of incubation. The ipsilaterally directed fibers can be first seen on the fifth day of incubation and their numbers appear to increase until about embryonic day 12. At this stage the projection involves substantially more fibers than at hatching and is also more extensive in its distribution; in fact, in its general organization (but not its size) it closely parallels the normal crossed retinofugal system, contributing fibers to essentially all the primary visual relay nuclei in the diencephalon and midbrain and to much of the optic tectum, where the densest projection is to its caudomedial aspect. During the second week of incubation there is also a small number of retinal fibers, which after crossing in the optic chiasm, recross the midline in the posterior and tectal commissures (and also in the tectal roof plate), before ending in the pretectal region of the ipsilateral side. In addition, there is a markedly aberrant projection from the retina into the contralateral optic nerve. Most of the ipsilateral retinal fibers are eliminated between the twelfth and sixteenth days of incubation, and by day 17 the ipsilateral projection is reduced to its mature form. The progressive reduction in the ipsilateral projection occurs at a time when it is known (from other studies) that there is an appreciable loss of retinal ganglion cells; but whether the reduction is due to neuronal death or to the selective elimination of ipsilateral axon collaterals remains to be determined. The existence of a significant ipsilateral retinofugal component early in development, probably accounts, in part, for the distinctive and persistent ipsilateral projection that occurs if one eye is removed during the first few days of incubation.

Mechanisms of striatal pattern formation conservation of mammalian compartmentalization

The striatum is composed of two neuroanatomically and neurochemically defined compartments, termed the patches and matrix. We compared this compartmentalization of the striatum in sections from the rat, rhesus monkey and human, in terms of (1) total striatal area, (2) the ratio of patch to matrix areas, (3) the number of patches and (4) the cross-sectional area of individual patches. Dense mu-opiate receptor binding and immunohistochemical staining for enkephalin were used as histochemical markers for the patch compartment and heavy immunostaining for calcium binding protein was used as a matrix marker. Analysis of coronal sections revealed that a relatively constant ratio of 15% patch to 85% matrix area is maintained in each species. The numbers of patches also remain relatively constant across species, despite a 19-fold increase in total striatal area from rat to human. The constant ratio of patch to matrix areas is maintained by an increase in the size of the individual patches. We hypothesize that the maintenance of a 15% patch to 85% matrix ratio in the striata of different mammalian species occurs through proportionate changes in the length of striatal neurogenesis and the numbers of striatal precursors in the ventricular zone, whereas the maintenance of average patch number is proposed to be a function of reciprocal connections with the substantia nigra and adhesive factors that are specific to patch cells.

Intracranial self-stimulation from the sulcal prefrontal cortex in the rat: The effect of 6-hydroxydopamine or kainic acid lesions at the site of stimulation

An electrode cannula system was used to elicit intracranial self-stimulation (ICSS) from the sulcal prefrontal cortex in rats to test the behavioral effects of local infusions of 6-hydroxydopamine (6-OHDA) or kainic acid (KA) into the brain area surrounding the electrode tip. In experiment I sulcal ICSS animals received injections of 6-OHDA with or without desipramine (DMI) pretreatment to block 6-OHDA uptake into noradrenergic (NA) terminals. Those animals that received DMI pretreatment were subsequently shown to have sustained sulcal cortical dopaminergic (DA) denervation while sulcal molecular layer NA systems were spared as revealed with glyoxylic acid-induced catecholamine histofluorescence. Those animals not receiving DMI pretreatment sustained near-complete denervation of both NA and DA sulcal cortical systems. Neither treatment had a lasting effect on sulcal ICSS suggesting that sulcal ICSS is not dependent on the presynaptic release or DA of NA into that brain area. In experiment II KA injections that lesioned neurons in sulcal cortical layers V and VI resulted in the abolition of sulcal ICSS for the duration of a 21 day postlesion trial period. These results suggest that activation of a descending corticofugal system originating in the sulcal cortex is responsible for the mediation of sulcal prefrontal cortical ICSS. This system was mapped by the selective silver impregnation of degenerating neural elements resulting from effective lesions.

Immunohistochemical localization of a brain isozyme of phospholipase C (PLC III) in astroglia in rat brain

Three distinct brain isozymes of phosphoinositide-specific phospholipase C (PLC) have been previously characterized: PLC I (150 kDa), II (145 kDa) and III (85 kDa). The present immunohistochemical study of rat brain localizes PLC III to atroglia, whereas previous studies have demonstrated the neuronal localization of PLC I and II.