Transneuronal uptake of horseradish peroxidase in the central nervous system of dipterous insects

Summary Horseradish peroxidase (HRP) applied to lesioned neurons in the retina and thoracic ganglia of the flies Musca, Calliphora and Drosophila labeled axon terminals, dendrites and perikarya of the severed neurons after anterograde or retrograde passage. In addition, HRP reaction product secondarily labeled intact neurons that are contiguous with injured nerve cells. In many cases labeling of optic lobe neurons remote from primarily filled ones was also seen (here called tertiary labeling). HRP labeling was extensive and both primarily and transneuronally filled neurons could be resolved in almost as much detail as Golgi-impregnated or cobalt-silver-labeled cells. Electron microscopy showed that in both primarily and secondarily filled neurons, reaction product was distributed diffusely in the cytoplasm.Transneuronal uptake of HRP was specific to certain types of neurons in the brain and thus displayed certain pathways. The pathways resolved by transneuronal labeling with HRP extend from the optic lobes to the thoracic ganglia and include visual neurons previously identified electrophysiologically and anatomically.Transneuronal HRP uptake, although believed to occur in vivo, could not be shown to be dependent on synaptic activity. Three other heme peptides tested were taken up by injured neurons, but showed no transneuronal labeling: lactoperoxidase, cytochrome c, and microperoxidase.     

Tangential organization of the infragranular fiber plexus in rat cerebral cortex

Cylindrical lesions (diameter 300-500 microns) were formed by poking needles into various parts of the cerebral cortex of adult albino rats. Degenerating axons were visualized in horizontal sections through the 'flattened' cortex using the silver impregnation method of Gallyas et al. [Stain Technol. 55: 291-297 (1980)] which stains degenerating axoplasm. The density and distribution of tangentially oriented axons were evaluated in the infragranular layers by TV image analysis. The sampling fields were concentrically arranged around the lesion at distances of 200, 400, 700 and 1,100 microns. The results indicate that the distribution patterns of degenerating (associational) axons covary with the cytoarchitectonic regions into which the lesions were placed. In the motor cortex, the majority of axons run in the antero-posterior direction. The density is generally lower around lesions in frontal regions than in parietal regions. The most extended degeneration was found around lesions near the border of or within the retrosplenial cortex, indicating an exceptionally strong internal connectivity in this area. Since only few degenerating axons were seen around lesions in the center of area 17, the high density of myelinated axons in the primary visual cortex seems to be due to fibers that originate in peristrate areas. It is concluded that the number and extension of fibers that degenerate tends to covary with some aspects of cortical architecture, but it is not area-specific.     

Visual thalamic afferents to area 29 of the rat limbic cortex

Afferent connections of the retrosplenial area of the rat limbic cortex were investigated by the retrograde horseradish peroxidase axon transport method. After injection of horseradish peroxidase (HRP) into area 29 of the cortex, HRP-labeled cells were found in the dorsal part of the lateral geniculate body and the posterolateral, pretectal, and anterior dorsal thalamic nuclei. Connections were found between cortical area 29 and visual projection areas (areas 17 and 18a) and with area 29 on the contralateral side of the brain. The results are evidence that all the principal visual structures of the thalamus and the visual cortical projection area form direct projections to the retrosplenial cortex.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 14, No. 2, pp. 135–139, March–April, 1982.     

Direct connections of the hippocampus with the retrosplenial cortex in rats

Connections of the retrosplenial cortex with the hippocampus in rats were investigated by the method of retrograde axonal transport of horseradish peroxidase (HRP). If a crystal of HRP was introduced into the dorsocaudal part of the retrosplenial cortex, HRP-labeled cells were found in the rostral pole of hippocampal area CA₃ and in the presubiculum. After iontophoretic injection of HRP into the rostral pole of the hippocampus, HRP-labeled cells were found in layers V and VI of the retrosplenial cortex and in the presubiculum. The results are evidence of reciprocity of direct connections between the rostral pole of the hippocampus and the retrosplenial cortex, and also of the existence of direct efferents of the dorsal subiculum to the rostral pole of the hippocampus.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 17, No. 1, pp. 102–107, January–February, 1985.     

5—HT能纤维向猫脊髓膈神经核及延髓膈肌前运动神经元的投射

实验在6只成年猫上进行,将WGA－HRP微量注入C5膈神经核内,通过逆行追踪及5－HT免疫组织化学FITC荧光双重标记方法,研究了中缝核5－HT能神经元向脊髓膈神经核的投射,同时观察了延髓膈肌产运动神经元接受5－HT能纤维投射的情况,结果在中缝苍白核观察到较多的HRP－5－HT双标记神经元,在中缝大核,中缝隐核观察到少数散在的双标记神经元,在延髓疑核,孤束核腹外侧区域的HRP单核记神经元（即膈肌前运动神经元）周围观察到5－HT能轴突末梢,结构表明：发自中缝苍白核5－HT能神经元的传出纤维可投射到脊髓膈神经核,延髓膈肌前运动神经元接受5－HT能纤维的传入投射。     

Modification of callosal afferents of the primary visual cortex ipsilateral to the remaining eye in rats monocularly enucleated at different stages of ontogeny

Summary Callosal afferents to the primary visual cortex (area Oc1) mainly originate in the border region between the lateral portion of the primary visual cortex (area Oc1) and the laterally positioned secondary visual cortex (area Oc2L) of the contralateral hemisphere. The extent of this region has been determined by retrograde labeling with horseradish peroxidase (HRP). In normal rats the width of the retrogradely labeled cortical strip is about 0.3 mm. In rats monocularly enucleated from the 23rd up to the 44th ontogenetic day and subsequently injected as adults with HRP into Oc1 ipsilateral to the remaining eye, the perikarya of the callosal afferents from the opposite hemisphere are labeled in the form of significantly wider columns (about 0.8 mm) than in animals enucleated from the 50th ontogenetic day onwards. The latter do not differ from controls.     

Glutamatergic components of the retrosplenial granular cortex in the rat

The ultrastructural characteristics, distribution and synaptic relationships of identified, glutamate-enriched thalamocortical axon terminals and cell bodies in the retrosplenial granular cortex of adult rats is described and compared with GABA-containing terminals and cell bodies, using postembedding immunogold immunohistochemistry and transmission electron microscopy in animals with injections of cholera toxin- horseradish peroxidase (CT-HRP) into the anterior thalamic nuclei. Anterogradely labelled terminals, identified by semi-crystalline deposits of HRP reaction product, were approximately 1 μm in diameter, contained round, clear synaptic vesicles, and established asymmetric (Gray type I) synaptic contacts with dendritic spines and small dendrites, some containing HRP reaction product, identifying them as dendrites of corticothalamic projection neurons. The highest densities of immunogold particles following glutamate immunostaining were found over such axon terminals and over similar axon terminals devoid of HRP reaction product. In serial sections immunoreacted for GABA, these axon terminals were unlabelled, whereas other axon terminals, establishing symmetric (Gray type II) synapses were heavily labelled. Cell bodies of putative pyramidal neurons, containing retrograde HRP label, were numerous in layers V–VI; some were also present in layers I–III. Most were overlain by high densities of gold particles in glutamate but not in GABA immunoreacted sections. These findings provide evidence that the terminals of projection neurons make synaptic contact with dendrites and dendritic spines in the ipsilateral retrosplenial granular cortex and that their targets include the dendrites of presumptive glutamatergic corticothalamic projection neurons.     

Connections of neurons in the lumbar ventral horn of spinal cord are altered after long-standing limb loss in a macaque monkey

Explanations for the massive reorganization in primary motor cortex, M1, after limb amputation typically focus on processes that occur in cortex. Few have investigated whether changes in more peripheral parts of the pathway might also play a role in the reorganization. In the present study, we examined the integrity and connectivity of the spinal cord motoneurons in a macaque monkey (Macaca mulatta) that lost a hindlimb as a result of accidental injury more than 3.5 years earlier. To label motoneurons, multiple small injections of a neuroanatomical tracer were placed in the muscles of the hip just adjacent to the stump of the amputated leg, and in matched locations in the opposite side for control purposes. Injections of a second tracer were made in the intact foot. In the ventral horn that related to the intact hindlimb, motoneurons labeled by the hip injections were concentrated rostral and ventromedial to those labeled by the foot injections. Hip injections on the side of the amputation labeled neurons that were located well beyond the normal territory for motoneurons related to the hip and into the zone normally occupied by neurons projecting to the foot. Labeled motoneurons innervating the intact limb were significantly larger than neurons on the side of the amputation (x = 2410 and 2061 microm(2), respectively). The findings suggest that many neurons survived the long-standing amputation, and made new connections with remaining intact muscles. These new patterns of connectivity likely contribute to the reorganization of motor cortex in amputees, and perhaps to abnormal behaviors like those reported by human amputees.     

Retrosplenial and hippocampal projections of structures of the thalamoparietal associative system in rats

Afferent connections of the nucleus lateralis posterior (NLP) of the thalamus and area 7 of the parietal cortex with the retrosplenial region of the limbic cortex and hippocampus were studied in rats with retrograde axon transport of horseradish peroxidase. It was shown that the NLP receives ipsilateral projections from area 29d neurons, while area 7 receives ipsilateral axons from area 29d and 29c neurons. It was found that associations of the retrosplenial region with associative cortex are far more pronounced than with associative thalamus. Moreover, the afferent connections of area 7 with area 29d are more numerous than with area 29c. We disclosed no projections of areas 29a and 29b to thalamoparietal system structures. In addition to neocortical input from the limbic cortex, area 7 receives afferent fibers from the archicortex; neurons situated in hippocampus area CA1 are the source of these projections.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy Academy of Sciences, Leningrad. Translated from Neirofiziologiya, Vol. 23, No. 6, pp. 647–655, November–December, 1991.     

Elimination of inhibitory synapses is a major component of adult ocular dominance plasticity

During development, cortical plasticity is associated with the rearrangement of excitatory connections. While these connections become more stable with age, plasticity can still be induced in the adult cortex. Here we provide evidence that structural plasticity of?inhibitory synapses onto pyramidal neurons is?a major component of plasticity in the adult neocortex. In?vivo two-photon imaging was used to monitor the formation and elimination of fluorescently labeled inhibitory structures on pyramidal neurons. We find that ocular dominance plasticity in the adult visual cortex is associated with rapid inhibitory synapse loss, especially of those present on dendritic spines. This occurs not only with monocular deprivation but also with subsequent restoration of binocular vision. We propose that in the adult visual cortex the experience-induced loss of inhibition may effectively strengthen specific visual inputs with limited need for rearranging the excitatory circuitry.     

Fast cortical oscillation after thalamic degeneration: pivotal role of NMDA receptor

We examined electrophysiological and molecular changes of the thalamocortical system after thalamic degeneration in Purkinje cell degeneration (pcd) mice. In pcd mice, neurons in specific thalamic nuclei including the ventral medial geniculate nucleus began to degenerate around postnatal day 50, whereas the visual thalamic nucleus and nonspecific thalamic nuclei remained almost intact. In association with the morphological changes, auditory evoked potentials in the primary auditory cortex (AC) began to decrease gradually. Fast Fourier transform analysis of spontaneous cortical field potentials revealed that fast oscillation (FO) around 25 Hz occurred in the AC but not in the visual cortex. Quantitative mRNA analysis demonstrated that expression of the N-methyl-D-aspartate (NMDA) receptor was up-regulated in the AC but not in the visual cortex. Systemic administration of an NMDA antagonist abolished the FO in the AC. These results indicate that increased NMDA activity may cause the FO in the AC of pcd mice.     

大鼠下丘脑室旁核与终纹床核及前额叶皮质间纤维联系的研究

分别注射辣根过氧化物酶（HRP）入大鼠的PVN和BNST,用组织化学的方法在确定注射部位准确的情况下,在PVN、BNST及PFC观察被标记的神经元或轴突末梢,探讨大鼠下丘脑室旁核（PVN）与终纹床核（BNST）及前额叶皮质间（PFC）之间是否存在投射通路;将HRP注射到PVN后,在同侧的BNST见标记的细胞体,在PFC未见标记的细胞体或轴突末梢;将HRP注射到BNST后,在同侧的PVN见标记的轴突末梢,在PFC未见标记的细胞体或轴突末梢。大鼠BNST有神经纤维投射到PVN,PFC与PVN及BNST之间没有直接的或只有极少量的纤维联系,在机体面临威胁性情境时,BNST可能激活HPA轴引发生理和行为反应,PFC是否通过与PVN或BNST的直接或间接的纤维投射实现其调节功能值得关注。     

大白鼠脚内核的传入性联系

众所周知,肉食动物和大白鼠的脚内核,相当于灵长类的内侧苍白球(Nagy et al.1978;Fox and Schmitz 1944);它们的细胞形态、传入及传出均相同。早期以及近年来的一些研究工作者,虽然在研究其他核团的投射时,联系到一些本核团的传入,但是尚缺乏对本核团传人的系统研究。本实验即是应用辣根过氧化物酶的逆行传递法来研究大白鼠脚内核的传入性联系。     

The temporo-spatial course of degeneration after cutting cortico-cortical connections in adult rats

Summary Adult albino rats received callosotomies or lesions in the paracingular cortex. Between 12 h and 3 months after injury the structure and topography of the degeneration products were studied by light- and electron-microscopy. The degeneration process was quantified by television-image analysis applied to sections prepared according to a new technique that stains reliably degenerating terminals and lysosomes (Gallyas et al. 1980). All types of cortico-cortical connections show a multiphasic degeneration process: During a precursor stage a small number of dense bodies and mitochondrial granules are stained. These and the few early degenerating axon terminals are much more diffusely distributed than the large number of terminals that degenerate during the following period. The terminal degeneration shows a biphasic time course. One maximum appears at 2–7 days post operation, which corresponds to the well known direct consequence of axotomy. The second peak at 10–20 days post operation could be caused by transneuronal reorganization of the cortical connectivity. Terminal degeneration always begins along the borders between cortical regions and areas, but it may change its laminar and columnar distribution pattern during the second phase. The degeneration products that are phagocytosed by astrocytes seem to be removed by intracellular transport to their perivascular endfeet. The degeneration process ends with fiber degeneration which, especially in laminae I and VI, may form a separate peak after 20 days or more.On leave from: Department of Neurosurgery, University of Göttingen, Federal Republic of Germany;On leave from: First Department of Anatomy, Semmelweis University, Medical School, Budapest, Hungary     

Experience with the use of horseradish peroxidase for light and electron microscope studies of interneuronal connections]

By the method based on a retrograde axonal transport of exogenous horseradish peroxidase (HRP), the origins of afferentation of the motor cortex of adult cats, kittens and albino rats were studied. HRP-positive neurons were found by light and electron microscopy in the somatosensory cortex (C1) of the ipsilateral hemisphere and in the portions of the cortex of the contralateral hemisphere which were symmetrical to the site of injection of HRP. The disposition of neurons, marked by HRP, in the Vth layer of the motor cortex suggest that these neurons may send their axons into the bundles of comissural fibres going to the motor cortex of the opposite hemisphere. This method considerably expands possibilities of revealing the origins of afferentation of the investigated portion of the nervous system and allows more complete and reliable investigation of interneuronal connections.     

Projections of Olfactory Bulbs and Non-Olfactory Telencephalic Structures in Amygdaloid Complex of the Turtle <Emphasis Type="Italic">Testudo horsfieldi</Emphasis>: A Study Using Anterograde Tracer Technique

To confirm the discrete character of projections of telencephalic olfactory and non-olfactory structures to the amygdaloid complex (AC) in the terrestrial turtle Testudo horsfieldi, a study was performed by the method of anterograde axonal transport of tracers (HRP, BDA). After a massive injection of the tracers into the main and accessory olfactory bulb, a dense accumulation of labeled fibers and terminals was found in ventral part of AC in the neuropil zones of nbam (J) and ncoam and very scanty—in nmam and ncam. After a massive injection of the tracers into non-olfactory telencephalic structures including dorsal cortex, pallidal enlargement, and ADVR, a very dense terminal field was observed in the dorsal AC part and a less dense one, with predominance of labeled fibers, in its ventral part. Local administration of the tracers separately into the dorsolateral (visual area) and the ventromedial (auditory-somatic area) parts of the ADVR allowed revealing discrete projections, respectively, to the laterocentral and mediocentral areas of the dorsal AC part with a relative overlapping in the central AC area. In all experiments, retrogradely labeled neurons in AC were also observed in zones of the corresponding bulbar and rostrotelencephalic projections. Thus, it has been shown that in the turtle AC there exist not only separation of direct olfactory and non-olfactory projections, but also discrete projections of different sensory areas of ADVR. Reciprocity of these connections is also confirmed. Organization of afferent olfactory and non-olfactory telencephalic connections in AC is similar in reptiles and in mammals.     

Areal Distributions of Cortical Neurons Projecting to Different Levels of the Caudal Brain Stem and Spinal Cord in Rats

Distributions of corticospinal and corticobulbar neurons were revealed by tetramethylbenzidine (TMB) processing after injections of wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP) into the cervical or lumbar enlargements of the spinal cord, or medullary or pontine levels of the brain stem. Sections reacted for cytochrome oxidase (CO) allowed patterns of labeled neurons to be related to the details of the body surface map in the first somatosensory cortical area (SI). The results indicate that a number of cortical areas project to these subcortical levels: (1) Projection neurons in granular SI formed a clear somatotopic pattern. The hindpaw region projected to the lumbar enlargement, the forepaw region to the cervical enlargement, the whisker pad field to the lower medulla, and the more rostral face region to more rostral brain stem levels. (2) Each zone of labeled neurons in SI extended into adjacent dysgranular somatosensory cortex, forming a second somatotopic pattern of projection neurons. (3) A somatotopic pattern of projection neurons in primary motor cortex (MI) paralleled SI in mediolateral sequence corresponding to the hindlimb, forelimb, and face. (4) A weak somatotopic pattern of projection neurons was suggested in medial agranular cortex (Ag_m), indicating a premotor field with a rostromedial-to-caudolateral representation of hindlimb, forelimb, and face. (5) A somatotopic pattern of projection neurons representing the foot to face in a mediolateral sequence was observed in medial parietal cortex (PM) located between SI and area 17. (6) In the second somatosensory cortical area (SII), neurons projecting to the brain stem were immediately adjacent caudolaterally to the barrel field of SI, whereas neurons projecting to the upper spinal cord were more lateral. No projection neurons in this region were labeled by the injections in the lower spinal cord. (7) Other foci of projection neurons for the face and forelimb were located rostral to SII, providing evidence for a parietal ventral area (PV) in perirhinal cortex (PR) lateral to SI, and in cortex between SII and PM. None of these regions, which may be higher-order somatosensory areas, contained labeled neurons after injections in the lower spinal cord. Thus, more cortical fields directly influence brain stem and spinal cord levels related to sensory and motor functions of the face and forepaw than the hindlimb.

The termination patterns of corticospinal and corticobulbar projections were studied in other rats with injections of WGA:HRP in SI. Injections in lateral SI representing the face produced dense terminal label in the contralateral trigeminal complex. Injections in cortex devoted to the forelimb and forepaw labeled the contralateral cuneate nucleus and parts of the dorsal horn of the spinal cord. The cortical injections also demonstrated interconnections of parts of SI with some of the other regions of cortex with projections to the spinal cord, and provided further evidence for the existence of PV in rats.     

Afferent connections in the ventromedial hypothalamus of rats demonstrated by retrograde transport of horseradish peroxidase

Projections into rat ventromedial hypothalamus were studied with retrograde transport of horseradish peroxidase (HRP). Following injection of HRP into ventromedial hypothalamus, labeled neurons were found in cortical and medial amygdaloid nuclei, ipsilateral mediodorsalis thalamus (MD), dorsal raphe nucleus, and contralateral sensorimotor cortex. Futhermore, labeled axons that connect directly amygdala with hypothalamus (DAH) also were found.     

Ovarian hormones after postnatal day 20 reduce neuron number in the rat primary visual cortex

Previous work from our lab has documented a sex difference in neuron number in the binocular region of the adult rat primary visual cortex (Oc1B), with males having 19% more neurons than females. In the present study, the role of developmental steroid hormones in the formation of this difference was explored. Male and female rats underwent neonatal hormone manipulation (female + testosterone or dihydrotestosterone; male + flutamide) followed by gonadectomy on postnatal day 20. Animals that did not undergo hormone manipulation were either gonadectomized or sham operated at day 20. Neuron number was quantified in the monocular (Oc1M) and binocular (Oc1B) subfields of the adult rat primary visual cortex using the optical disector technique. As adults, day 20 gonadectomized females, as well as females + testosterone and females + dihydrotestosterone, had significantly more neurons than intact females. There was no difference in neuron number between postnatal day 20 gonadectomized males, males + flutamide, and intact males. Also, intact males had significantly more neurons than intact females in both in Oc1M and Oc1B. It appears that ovarian steroids after day 20 are the primary cause of the lower number of neurons in the primary visual cortex of the female rat.     

Multisynaptic Inputs from the Medial Temporal Lobe to V4 in Macaques

Retrograde transsynaptic transport of rabies virus was employed to undertake the top-down projections from the medial temporal lobe (MTL) to visual area V4 of the occipitotemporal visual pathway in Japanese monkeys (Macaca fuscata). On day 3 after rabies injections into V4, neuronal labeling was observed prominently in the temporal lobe areas that have direct connections with V4, including area TF of the parahippocampal cortex. Furthermore, conspicuous neuron labeling appeared disynaptically in area TH of the parahippocampal cortex, and areas 35 and 36 of the perirhinal cortex. The labeled neurons were located predominantly in deep layers. On day 4 after the rabies injections, labeled neurons were found in the hippocampal formation, along with massive labeling in the parahippocampal and perirhinal cortices. In the hippocampal formation, the densest neuron labeling was seen in layer 5 of the entorhinal cortex, and a small but certain number of neurons were labeled in other regions, such as the subicular complex and CA1 and CA3 of the hippocampus proper. The present results indicate that V4 receives major input from the hippocampus proper via the entorhinal cortex, as well as “short-cut” pathways that bypass the entorhinal cortex. These multisynaptic pathways may define an anatomical basis for hippocampal-cortical interactions involving lower visual areas. The multisynaptic input from the MTL to V4 is likely to provide mnemonic information about object recognition that is accomplished through the occipitotemporal pathway.