P. Somogyi

An approach to tracing neuron networks in the cerebral cortex and basal ganglia. Combination of Golgi staining, retrograde transport of horseradish peroxidase and anterograde degeneration of synaptic boutons in the same material.

Abstract A procedure is described that makes it possible to study, in the light microscope and in the electron microscope, neurons that have been labelled by the retrograde transport of horseradish peroxidase in brain tissue that is also subsequently stained by the Golgi method. The procedure involves the use of o -tolidine (3,3′-dimethylbenzidine) as a substrate for horseradish peroxidase; the reaction end product from this substrate can be formed in blocks of tissue of the size needed for the Golgi method and can be recognized both in the light and in the electron microscope. In order to detect the reaction end product within Golgi-stained neurons in the light microscope, the Golgi stain is replaced by gold (‘gold-toning’). It then becomes possible to identify the type of efferent neuron (in an area we call B), on the basis of its characteristic shape and dendritic morphology, that projects to the area (C) where horseradish peroxidase was injected. Furthermore, the afferent input to such a characterized efferent neuron can be studied if a lesion had previously been placed in a third area (A) that projects to area B; degenerating terminal boutons making contact with any part of the Golgi-stained, horseradish peroxidase labelled neuron can then be identified in the electron microscope. In this way, the existence of a chain of neurons within a network can be established starting with a neuron in area A, through a monosynaptic link to a neuron (in area B) that sends a projection to area C. In addition, if the region that is stained by the Golgi method contains Golgi-stained local circuit interneurons, it is possible to study the input from these interneurons to an efferent neuron that has been labelled retrogradely with horseradish peroxidase. The procedure is illustrated by three specific applications that have provided the following new information concerning connections in the brain of the rat. (i) Some of the neurons that project from the striatum to the substantia nigra are medium sized and have densely spinous dendrites. (ii) One type of striatonigral neuron terminates (symmetrical synapses) on smooth dendrites of nigrothalamic neurons in the substantia nigra pars reticulata. (iii) A specific type of local circuit interneuron (the ‘chandelier cell’ or ‘axo-axonic’ cell) in the visual cortex terminates (symmetrical synapses) on the axon initial segments of pyramidal cells that project to the contralateral visual cortex. Some possible functional implications of these findings are discussed. It is suggested that further applications of the procedure might provide direct information about many aspects of synaptic connectivity in neuron networks in parts of the central nervous system that are too complex to study by conventional morphological methods.

The axo-axonic interneuron in the cerebral cortex of the rat, cat and monkey

The synaptic connections of a specific type of identified cortical interneuron, the axo-axonic cell, were studied using Golgi methods. In the light-microscope axo-axonic cells were demonstrated in certain layers of the primary and secondary visual cortex of rat, cat and monkey, in the motor cortex of cat and in the subiculum and pyriform cortex of rat. The dendrites originating from the oval soma were oriented radially in a lower and upper spray within a cylinder about 100-150 microns wide. Electron-microscopy of Golgi impregnated, gold-toned axo-axonic cells showed predominantly but not exclusively asymmetrical synaptic contacts on their dendrites and spines, few synaptic contacts on the perikarya some of which were asymmetrical, and no synaptic contacts on the axon initial segment. The axon usually arborized within the vicinity of the cells own dendritic field in an area 100-200 microns in diameter. In the kitten motor cortex the axon of a neuron in layer III descended to layer VI, providing a columnar arborization. The axon formed specialized, 10-50 microns long terminal segments invariably oriented parallel with the axon initial segment of pyramidal cells. All 85 identified symmetrical-type synaptic contacts, deriving from 31 specialized terminal segments, were found exclusively on the axon initial segment of pyramidal neurons. Rare, lone boutons of axo-axonic cells also made synaptic contact only with axon initial segments, confirming the exclusive target specificity of these cells. In identified gold-toned boutons, flattened pleomorphic vesicles were present. Electron-microscopy showed that axons ending in specialized terminal segments may originate from myelinated fibres, indicating that Golgi impregnation has revealed only part of the axon. Counting of axon terminal segments, each of which was in contact with the axon initial segment of a pyramidal neuron, revealed 166 pyramidal neurons receiving input from a partially reconstructed axo-axonic cell in the motor cortex of the kitten, and 67 from another cell in the visual cortex of the cat. The convergence of five axo-axonic cells onto one pyramidal cell was demonstrated in the striate cortex of the cat by counting all synaptic contacts on three initial segments. Cells from a one-month-old kitten were compared with those of the adult. The axon of the developing neurons was more diverse, having many growth cones and filopodia which made no specialized membrane contacts. However, the developing specific terminal segments formed synapses only with axon initial segments. It is concluded that the presence of axo-axonic cells in all the species and cortical areas we have examined suggests their association with the structural design of pyramidal cells, wherever the latter occur, and with their participation in the information processing of pyramidal cells. Axo-axonic cells are uniquely endowed with the means of simultaneously influencing the action potential at the site of origin in groups of pyramidal cells...

Monosynaptic input from the nucleus accumbens-ventral striatum region to retrogradely labelled nigrostriatal neurones

After placement of lesions (either electrolytic or by injection of kainic acid) in an area including the nucleus accumbens and part of the ventral striatum in the rat, the ipsilateral substantia nigra was studied in the electron microscope. Degenerating axons and nerve terminals were found mainly in the zona reticulata and in the ventral layer of the zona compacta. Degenerating synaptic boutons were found in contact with cell bodies (symmetric synapses) and dendrites (mainly symmetric, but a few asymmetric). The postsynaptic target of some of the afferent fibres from the accumbens-ventral striatum was established by demonstrating degenerating synaptic boutons of the above types in contact with nigrostriatal neurones which had been identified by the retrograde transport of horseradish peroxidase (HRP) from the main body of the striatum. Some of the HRP-labelled cells were also impregnated by the Golgi stain and degenerating boutons were found in contact with their distal dendrites. We also observed two types of HRP-containing boutons (presumably labelled anterogradely) in the subsantia nigra after injection of HRP into the main body of the striatum : type 1 boutons contained large spherical vesicles, and formed symmetrical synapses mainly on dendritic shafts in the zona reticulata and in one case the dendrite was from a nigrostriatal neurone; type 2 boutons had pleomorphic and flattened vesicles and formed symmetrical synapses with perikarya and proximal dendrites, especially in the zona compacta. The latter type of HRP-labelled bouton was frequently found in synaptic contact with the cell bodies of nigrostriatal neurones and the same neurones sometimes also received degenerating boutons originating from neurones in the nucleus accumbens-ventral striatum. It is concluded that part of the striato-nigro-striatal circuit includes a monosynaptic link between neurones in the ventral striatum-accumbens and some nigrostriatal neurones. The possible convergence of input from different regions of the striatum on to single nigrostriatal neurones is also suggested.

Projection of neostriatal spiny neurons to the substantia nigra. Application of a combined golgi-staining and horse-radish peroxidase transport procedure at both light and electron microscopic levels

One type of striatonigral neuron in the rat has been characterized. Golgi impregnation of striatal neurons that had been retrogradely labeled by horseradish peroxidase has shown that the medium-sized, densely spiny neurons project to the substantia nigra. Some of the synapses on three of these identified striatonigral neurons have been studied in the electron microscope following replacement of the Golgi deposit by means of the gold-toning method. Synapsing axonal boutons were found on the following sites: soma and axon initial segment (symmetrical, with flattened or pleomorphic vesicles); primary and secondary dendritic shafts (symmetrical with pleomorphic vesicles); dendritic spines (asymmetrical, with spheroidal vesicles). These findings show that new information concerning neuronal connectivity can be obtained by combining three classical procedures in the same material: first, the Golgi method, that characterizes the type of neuron on the basis of its dendritic morphology; second, a retrograde tracing method, that identifies the projection area of the neuron; and, third, ultrastructural analysis of the nature of afferent terminals on the neuron.

Localization of substance P-like immunoreactivity in neurons and nerve terminals in the neostriatum of the rat: a correlated light and electron microscopic study.

SummaryAn antiserum, to substance P has been used to study the neostriatum of rats which has received intracerebral injections of colchicine. Both cell bodies and nerve fibres were found to display immunoreactivity. Some of the fibres were swollen and could be traced back to their parent: cell body.Examination in the electron microscope of structures that had first been identified in the light microscope showed that there are two different types of substance P-immunoreactive cell body. The first kind (type I) of immunoreactive cell body was of medium size and had a smooth surfaced nucleus. It displayed the ultrastructural features typical of medium-size spiny neurons. Identified axons of type I neurons gave rise to immunoreactive axon collaterals within the neostriatum boutons along these collaterals were found to form symmetrical synaptic contacts. The second kind (type II) of immunoreactive cell body was also of medium-size and had a round or oval shape, but the nucleus was deeply indented and was surrounded by a thin rim of cytoplasm. Synaptic input to this neuron was sparse and consisted of small boutons that made symmetrical contacts with the perikaryon and proximal dendrites.Many immunoreactive dot-like structures could be seen in the light microscope: upon examination in the electron microscope these were found to be boutons. All fifty-six synaptic boutons that were studied made symmetrical synaptic contacts. These boutons were indistinguishable from the boutons of axon collaterals of identified type I immunoreactive neurons. The most common postsynaptic structures were dendrites, including some dendritic spines, although synapses between immunoreactive boutons and several perikarya, and an axon initial segment were observed. The morphological features of the immunoreactive boutons in the neostriatum were very similar to one type of substance P-immunoreactive bouton in the substantia nigra and to a bouton type in the substantia nigra which is labelled following the anterograde transport of horseradish peroxidase from the striatum.It is suggested that there are two kinds of substance P-containing neurons in the striatum and that one of these is likely to belong to the medium-spiny class. The latter type of neuron is probably the source of the striatonigral substance P-containing projection and of the immunoreactive boutons within the striatum. The finding of substance P-immunoreactive synaptic boutons within the neostriatum provides a morphological basis for the view that substance P might serve as a neurotransmitter in the neostriatum.

Physiological and morphological properties of identified basket cells in the cat's visual cortex

SummaryIn 87 cells studied physiologically, and filled intracellularly with horseradish peroxidase (HRP), we have found four cells which make multiple contacts with the perikarya of their post-synaptic targets. These cells are all multipolar non-pyramidal neurones with elongated smooth dendrites. Three resemble the classical “basket cells” of Ramón y Cajal (1911), having widely distributed axons which contribute to the “nids pericellulaires” around pyramidal cell perikarya. The fourth cell has a much more restricted axon virtually confined to layer 4 and appears to contact principally small, probably nonpyramidal, cells. Two of the basket cell axons have been examined by electron microscopy and make symmetrical, Grays type II contacts with the perikarya and apical and basal dendrites of pyramidal cells. Ten percent of the synapses are on dendrites of non-pyramidal cells.The axon arborizations of all four cells are distributed in a patchy fashion. In two cells examined for the purpose, very few boutons were found within 100 μm. of the cell body and a radially aligned cylinder of the same diameter extending from the cell body to the pial surface. The physiological properties of these structurally similar cells are far from uniform. They can be activated mono- or polysynaptically, by X- or Y-type lateral geniculate input, and can have S or C type receptive fields. Two were activated, probably monosynaptically, via callosal afferents. These cells may play an important role in the inhibitory mechanisms of the cortex.

The study of Golgi stained cells and of experimental degeneration under the electron microscope: a direct method for the identification in the visual cortex of three successive links in a neuron chain.

Abstract A direct method is presented which makes it possible to identify, from synapse to synapse, three successive links of a neuron chain. The potentialities of the method are shown by examples of the termination of specific afferents in the visual cortex. Following unilateral lesion of the lateral geniculate nucleus in the rat, the distribution of degenerating geniculocortical boutons was studied on two Golgi-stained cells in layer IV of the primary visual cortex. One of the cells was definitely a small pyramidal cell; the other was identified as a spiny stellate (although the possibility that it too was a small pyramidal cell was not rigorously excluded). Both cells received monosynaptic input from the specific afferents as proved by the existence of degenerating boutons synapsing on their dendritic spines. The axonal arborizations of both Golgi-stained cells were traced at the electron microscopic level in thin section series in order to identify the postsynaptic structures contacted by their boutons. All boutons studied established asymmetrical contacts and about 50% of the synapses given by the impregnated boutons were found on smooth dendritic shafts of stellate cells, the rest on spines. The results obtained suggest a neuron circuit involving, successively, the visual afferents, spiny interneurons or monosynaptic visual target pyramidal cells and nonspiny stellate cells. It is suggested that a similar approach might provide direct information about the connectivity in neuron networks in many other parts of the central nervous system hitherto defying elucidation with conventional methods.

Synaptic connections of enkephalin-immunoreactive nerve terminals in the neostriatum: a correlated light and electron microscopic study

SummaryTwo different antisera to leucine-enkephalin were used to study the localization of enkephalin-like immunoreactive material in the neostriatum and globus pallidus of the rat, by means of the unlabelled antibody-enzyme method. Thin immunoreactive varicose fibres are scattered throughout the neostriatum. In the ventral striatum, fibres come together and follow a relatively straight course for several micrometers, forming tube-like structures which can be traced to cell bodies; these cell bodies are completely surrounded by immunoreactive fibres. Occasional immunoreactive varicose fibres are also found close to another type of neuron throughout the whole neostriatum.Examination by electron microscopy of immunoreactive structures that had been identified first in the light microscope, showed that each of the nearly 200 varicosities examined was a vesicle-containing bouton that formed a synaptic contact. Rarely were asymmetrical synaptic contacts found between immunoreactive boutons and dendritic spines. All other synapses formed by enkephalin-immunoreactive boutons were symmetrical. Two types of postsynaptic neuron were identified; the first type was a medium-sized neuron with the ultrastructural features of a typical striatal spiny neuron. The second type had a larger perikaryon surrounded by numerous immunoreactive varicosities that were found to be boutons forming symmetrical synapses. The long dendrites of this second type of neuron likewise received a dense input of immunoreactive boutons forming symmetrical synapses; such ensheathed dendrites were found to be the tube-like structures seen in the light microscope. The ultrastructural features of these neurons, notably a highly indented nucleus, were those of a rare type of striatonigral neuron. In the globus pallidus, all the enkaphalin-immunoreactive boutons studied formed symmetrical synapses with ensheathed dendrites and perikarya that were similar to the latter type of postsynaptic neuron in the neostriatum. Axo-axonic synapses involving immunoreactive boutons were not seen in our material.The results are consistent with the view that enkephalin-like substances may be synaptic transmitters in the neostriatum and that they may have different actions according to the nature of the postsynaptic target. The finding that one type of neostriatal neuron, and a very similar neuron in the globus pallidus, receives multiple enkephalin-immunoreactive boutons all over its perikaryon and along its dendrites indicates a potentially important role of enkephalin in the convergence of information within the neostriatum and pallidum on to output neurons.

A second type of striatonigral neuron: a comparison between retrogradely labelled and golgi-stained neurons at the light and electron microscopic levels

Abstract In a light and electron microscopic examination of the neostriata of rats that had received injections of horseradish peroxidase into the ipsilateral substantia nigra, two morphologically distinct types of horseradish peroxidase-labelled neurons were observed. In confirmation of previous findings, one type was of medium-size and was characterized by Golgi-staining and gold-toning as the densely spinous type. The second type of neuron was in contrast, larger, had an indented nucleus and numerous cytoplasmic organelles. The synaptic input to the perikarya of the latter neurons consisted of numerous boutons containing large round and oval vesicles. The boutons formed symmetrical synaptic contacts and were similar to those of the local axon collaterals of medium-size densely spiny striatonigral neurons. In an attempt to establish what type of Golgi-impregnated neuron the second type of horseradish peroxidase-labelled neuron was, seventeen Golgi-stained or gold-toned neurons were examined in the electron microscope. Three of them were very similar in their ultrastructural features and synaptic input to the horseradish peroxidase-labelled neurons. All three were of a similar morphological appearance in the light-microscope and characteristically had long (up to 700 μm), essentially smooth dendrites. Both the large horseradish peroxidase-labelled neurons and the Golgi-impregnated neurons with long dendrites have so far only been found in the most ventral regions of the neostriatum. It is concluded that there are at least two morphologically distinct types of striatonigral neurons.

A possible structural basis for the extracellular release of acetylcholinesterase.

The ultrastructural localization of acetylcholinesterase and non-specific cholinesterase activity has been studied in sections of ox adrenal medulla by cytochemical methods. Non-specific cholinesterase activity, identified by using butyrylthiocholine as substrate and ethopropazine as inhibitor, occurs intracellularly in some adrenaline-containing chromaffin cells: the reaction end-product is deposited within the cisternae of the endoplasmic reticulum and in the nuclear envelope. Reaction end-product of non-specific cholinesterase also occurs in the endoplasmic reticulum of pericytes, around sinusoids and capillaries and within smooth muscle cells. Acetylcholinesterase activity, identified by using acetylthiocholine as substrate and BW 284C51 as inhibitor, occurs in both the splanchnic nerve and in chromaffin cells. Reaction end-product is found at the following sites (i) around myelinated and unmyelinated non-terminal axons of splanchnic nerve, between the axolemma and the Schwann cell membrane; (ii) within the cisternae of axonal smooth endoplasmic reticulum; sometimes these cisternae appear to be connected to the axolemma; (iii) between the axolemmas of preterminal axons and the plasma membranes of chromaffin cells; (iv) between the axolemmas of nerve terminals and the plasma membranes of chromaffin cells, including the synaptic cleft; (v) within cisternae of rough and smooth endoplasmic reticulum, and also within the nuclear envelope, of both adrenaline- and noradrenaline-containing chromaffin cells; (vi) between the plasma membranes of adjacent chromaffin cells, but only when one or both of these cells contain reaction product within the cisternae of its endoplasmic reticulum; these cisternae sometimes appear to be connected to the plasma membrane. These observations raise the question whether the acetylcholinesterase activity released from the perfused adrenal gland might originate from the cisternae of the endoplasmic reticula of splanchnic nerve and/or chromaffin cell.