Tetsuo Sugimoto

Application of coupled oxidation reaction to electron microscopic demonstration of horseradish peroxidase: cobalt-glucose oxidase method

Since the horseradish peroxidase (HRP) method was first applied to the peripheral nervous system by Kristensson and Olsson 12 and subsequently to the central nervous system by LaVail and LaVai116, it has become one of the most common experimental methods for demonstrating neuronal connectivity in the nervous system (for review see refs. 10 and 15). Previous studies have shown that the light microscopical procedure for demonstrating HRP can also be used for electron microscopical visualization of the enzyme, not only of the enzyme transported retrogradely to neuronal somata and dendrites3,5,8,1a,14,17,19,26, 28, but also of that transported anterogradely to axon terminals2,4,s,9,17,22-za,z6, zS. In an attempt to increase the sensitivity of the HRP method and to improve the reliability of tracing neuronal connections using retrograde and anterograde transport of HRP in combination with electron microscopy, the cobalt method of Adams 1 and the coupled oxidation reaction utilized by Lundquist and Josefsson 18 for the determination of low levels of tissue peroxidase were applied to the histochemical visualization of HRP. The coupled oxidation method, utilizing a continuous supply of hydrogen peroxide formed in the glucose oxidase (GOD) reaction, has been shown to be at least I0 times more sensitive than other colorimetric peroxidase assays 18, and also to be applicable to electron microscopical demonstration of HRP 27. In the present study, the coupled oxidation method, when applied to the histochemical demonstration of HRP in combination with cobalt method 1, was found to give excellent electron microscopical pictures of the electron dense reaction product. The experiments were performed in more than 50 adult cats anesthetized with i.p. Nembutal (35 mg/kg). For observation of the retrograde transport of HRP, 0.1-0.5 /~1 of 2 5 ~ HRP (Toyobo Grade-I-C, RZ: 3.4) was injected, in different experiments, into the masticatory muscles 2z, perineal muscles H, or wall of the urinary bladder 25. For detection of HRP transported anterogradely to axon terminals, HRP was injected in a variety of brain areas such as the cerebral cortical areas 21 and the motor trigeminal nucleus23; a single injection of 0.01~3.1 ~1 of 50 % HRP dissolved in

An autoradiographic study on the terminal distribution of cerebellothalamic fibers in the cat

Distribution of cerebellothalamic fibers was studied in the cat by the anterograde tracing method. The vast majority of cerebellothalamic fibers were distributed contralaterally. The fastigial fibers arose mostly from the caudal half of the nucleus. These ended moderately in the VM and the most ventromedial regions of the VA-VL complex, and sparsely in the ventral portions of the CL, Pc and NCM; a few also ended in the CM and ZI. The dentate fibers ended moderately in the median and rostrodorsal VA-VL regions, and sparsely in the VM, CL, NCM, CM, LP and MD. The existence of the dentatopulvinar fibers was also confirmed. The posterior interpositus fibers ended heavily in the central VA-VL regions, moderately in the subparafascicular nucleus and ZI, and sparsely in the CM and the ventral lateral geniculate nucleus. The anterior interpositus fibers ended mainly in the ventrolateral VA-VL regions, and additionally in the CL and CM. The ipsilateral cerebellothalamic fibers arose mainly from the fastigial nucleus, and additionally from the dentate nucleus; those arising from the interpositus nuclei were very sparse.

Localization of parabrachial nucleus neurons projecting to the thalamus or the amygdala in the cat using horseradish peroxidase

Abstract Topographical localization of parabrachial nucleus (PBN) neurons projecting directly to the thalamus or the amygdala was examined in the cat by the horseradish peroxidase (HRP) method. After HRP injection in the central nucleus of the amygdala, PBN neurons labeled with the enzyme were seen ipsilaterally in the ventral portion of the lateral PBN as well as in the medial PBN. When the HRP injections were centered on the parvocellular portion of the posteromedial ventral nucleus of the thalamus (VPMpc), HRP-labeled neurons were observed ipsilaterally in the dorsal portion of the lateral PBN as well as in the medial PBN. Within the medial PBN, the distribution of neurons projecting to the amygdala overlapped that of neurons projecting to VPMpc; the cell bodies of the former neurons, however, tended to be more elongated than the latter, and the mean of the average soma diameters of the former was significantly larger than the latter. On the other hand, in the lateral PBN no significant differences were noted between the means of the average soma diameters of neurons projecting to VPMpc and those projecting to the amygdala. The PBN neurons in the cat were presumed to transmit gustatory and general visceral information ipsilaterally to the thalamic taste region and the limbic areas in the basal forebrain.

Direct projections from the pedunculopontine tegmental nucleus to the subthalamic nucleus in the cat

The feline entopeduncular nucleus (EPN), the homologue to the internal segment of the pallidum in the primates, was currently shown to send fibers ipsilaterally to the mesencephalic tegmental regions around the brachium conjunctivum, particularly to the pedunculopontine nucleus3,12,1a or the nucleus tegmenti pedunculopontinus pars compacta (PPT) 15. The fibers corresponding to the feline EPN-PPT fibers were also reported to exist in the monkey 5,~,1s and the ratL As the EPN is the major output from the basal ganglia, further analysis of the EPN-PPT system is considered quite important in understanding the function of the basal ganglia. In addition to the afferent fibers from the EPN, the PPT reportedly receives afferent fibers from the substantia nigraT, 8, the cerebral cortex6, z0, the nucleus accumbens septi 19, and the subthalamic nucleus (STN) 16, but no conclusive data exist about the efferent connections of the PPT (for review, of. ref. 17). In this communication, a direct projection from the PPT to the STN in the cat will be described on the basis of the findings obtained by the autoradiographic labeling technique and the horseradish peroxidase (HRP) method. Microinjections of tritiated amino acids or HRP were conducted stereotaxically with a 1/~1 Hamilton microsyringe on 7 adult cats anesthetized with i.p. sodium pentobarbital (35 mg/kg); injection periods ranged from 10 to 20 min. In 4 cats, a single injection of 0.1--0.2/tl (40 #Ci//A) of equal parts L-[2,3-3H]proline and L-[4,5-aH] leucine (New England Nuclear) was made into the PPT. After 3-9 days of survival the cats were deeply anesthetized and perfused through the ascending aorta with 2 liters of 10 ~ neutral formalin. The brains were removed, frozen and cut at 40/~m in the frontal plane. Every fourth section was mounted onto gelatinized slides. These slides were then processed according to the standard autoradiography method 2 using Kodak

Distribution of cerebellar fiber terminals in the midbrain visuomotor areas: An autoradiographic study in the cat

Cerebellar fibers to the midbrain visuomotor areas were traced in the cat auto-radiographically after injections of tritiated amino acids into individual cerebellar nuclei. Fibers from the dentate (DN), anterior interpositus (AIN) and posterior interpositus (PIN) nuclei were distributed contralaterally, while those from the fastigial nucleus (FN) bilaterally. The FN fibers appeared to arise mainly from the caudal half of the FN. In the superior colliculus (SC), the FN or DN fibers were more numerous than the PIN fibers, and the areas of termination of the FN fibers were located more medially than those of the DN and PIN fibers. These cerebellotectal fiber terminals were in the intermediate and deep SC layers; clustering of terminal silver grains was noted in the FN and DN fibers-recipient areas in the intermediate gray layer. In the pretectum, the DN fibers terminated ventrally in the reticular part of the anterior pretectal nucleus and the posterior pretectal nucleus. THe AIN fibers terminated ventrally in the compact part of the anterior pretectal nucleus and the posterior pretectal nucleus. The nucleus of the posterior commissure received cerebellar fibers chiefly from the DN, and additionally from the FN. The nucleus of Darkschewitsch and the interstitial nucleus of Cajal received fibers from all cerebellar nuclei. No cerebellar fibers terminated in the extraocular motor nuclei and the Edinger-Westphal and anteromedian nuclei.

Topographical projections from the posterior thalamic regions to the striatum in the cat, with reference to possible tecto-thalamo-striatal connections

SummaryProjections from the posterior thalamic regions to the striatum were studied in the cat by the anterograde tracing method after injecting wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) into the caudalmost regions of the lateroposterior thalamic nucleus (caudal LP), suprageniculate nucleus (Sg) and magnocellular division of the medial geniculate nucleus (MGm). The results were further confirmed by the retrograde tracing method after injecting WGA-HRP into the regions of the caudate nucleus (Cd) and putamen (Put) where afferent fibers from the caudal LP, Sg and MGm were distributed. Fibers from the MGm, Sg or caudal LP were distributed mainly in the medial, middle or lateral part of the caudal half of the putamen (caudal Put), respectively. Although there was a considerable overlap, thalamostriatal fibers from the caudal LP terminated more caudally than those from the MGm. On the other hand, thalamocaudate fibers from the MGm, Sg and lateral part of the caudal LP overlapped with each other in the ventrolateral part of the caudal half of the caudate nucleus (caudal Cd). Fibers from the medial part of the caudal LP were distributed in the ventral part of the caudal Cd. In the superior colliculus (SC) of the cats with WGA-HRP injections in the caudal LP, retrogradely labeled neuronal cell bodies were mainly seen ipsilaterally in the superficial SC layer, and simultaneously, anterogradely labeled axon terminals were observed in the striatum. On the other hand, when WGA-HRP was injected into the Sg or MGm, labeled SC neurons were mainly located in the intermediate and deep SC layers. Thus, ascending impulses from the superficial SC layer may possibly be conveyed ipsilaterally via the caudal LP to the ventral and ventrolateral parts of the caudal Cd and the lateral part of the caudal Put, whereas those from the intermediate and deep SC layers may be relayed via the Sg and/or MGm to the ventrolateral part of the caudal Cd and the middle and medial parts of the caudal Put.

The site of origin of cardiac preganglionic fibers of the vagus nerve: An HRP study in the cat

After injection of horseradish peroxidase (HRP) into the right cardiac branches of the vagus nerve in the cat, the majority of HRP-labeled neurons were located ipsilaterally in the reticular formation ventrolateral to the nucleus ambiguus. Additionally, HRP-labeled neurons were also observed within the nucleus ambiguus (Am) and the dorsal motor nucleus of the vagus nerve (DM).

Direct projections from the Ediger-Westphal nucleus to the cerebellum and spinal cord in the cat: An HRP study

Abstract A vast majority of neurons in the Edinger-Westphal nucleus (EW), consisting of the anteromedian nucleus (AM) and the posterior part of the EW (EWp: so-called visceral nucleus), were labeled retrogradely with horseradish peroxidase (HRP) injected into the cerebellum in the cat. The pattern of distribution of these HRP-labeled EW neurons was similar to that of EW neurons labeled with HRP injected into the spinal cord. A tendency, however, was noted that EW neurons located in the nuclear areas close to the midline remained unlabeled in the cats injected with the enzyme into the spinal cord. It was assumed that some EW neurons might contribute fibers by way of axon collaterals both to the cerebellum and to the spinal cord. Possible function of the EW as a relay nucleus for retinal inputs to the cerebellum and the spinal cord was discussed.

Extrageniculate projections to the visual cortex in the macaque monkey: an HRP study.

Extrageniculate projections to the visual cortex were examined in the macaque monkeys by the horseradish peroxidase (HRP) method. Extrageniculate neurons sending fibers to the visual cortex were found in the lateral and inferior pulvinar nuclei, paracentral thalamic nucleus, claustrum, basal nucleus of Meynert, lateral part of the basal amygdaloid nucleus, lateral hypothalamus, locus coeruleus, and dorsomedial and midline regions of the pontine tegmentum.

Localization of neurons giving rise to the oculomotor parasympathetic outflow: A HRP study in cat

Abstract Localization of neurons giving rise to preganglionic fibers to the ciliary ganglion was attempted in the cat, utilizing retrograde axonal transport of horseradish peroxidase (HRP). After injection of HRP into the oculomotor nerve root at the level of the interpeduncular fossa, a few neurons of the Edinger-Westphal nucleus (EW) were labeled with HRP rostrally within the anteromedian nucleus (AM);HRP-labeled EW-neurons were rarely seen caudally within the visceral nucleus (VN). Other possible preganglionic neurons labeled with HRP were distributed mainly in rostromedial tegmental areas close to the lateral border of the AM, and in rostroventral areas of the mesencephalic central gray.