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

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 pm), 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. FROM the numerous Golgi studies that have been performed on the mammalian neostriatum, there is general agreement that there are up to six morphologically distinct types of neuron. These neurons have been classified into two major groups, medium-size and large, and further subdivided according to the presence or absence of dendritic spines and the arrangement of dendrites. The most frequently impregnated neuron is the medium-size densely spinous type, the remainder comprising medium-size and large neurons with sparsely spiny or smooth dendrites and a population of small neurons (for review see PASIK, PASIK & DI FIGLIA, 1979 ; see also DANNER & PFISTER, 1979; DIMOVA, VUILLET & SEITE, 1980). The major projection areas of neostriatal efferents are the globus pallidus and the substantia nigra ; attempts to correlate the type of projection neurons with Golgi-stained neurons have led to conflicting opinions. Thus, LEONTOVICH (1954) considered that the medium-size neurons with densely spinous dendrites are efferent cells while the classic views of the Vogts (VOGT & VOGT, 1920), supported by KEMP & POWELL (19710) and Fox, ANDRADE, HILLMAN & SCHWYN (1971), were that these neurons are local circuit neurons and that the large neurons are the efferent neurons of the neostriatum. Any conclusions drawn from Golgi material must, however, be tentaAbhreviations : GABA, y-aminobutyrate, HRP, horseradish peroxidase, STN, striatonigral neuron. 2141 tive since, unless a Golgi-stained axon is traced outside the striatum, it cannot be stated with certainty that the neuron projects. Even if the axon is traced outside the neostriatum, the target area of the neuron cannot be determined unless an axonal aborization is observed. A different approach to the study of efferent neurons is the labelling of cell somata following the retrograde transport of substances taken up at the axon terminals. In a study of the striatal and pallidal neurons projecting to the substantia nigra using the retrograde transport of horseradish peroxidase (HRP), GROFOVA (1975) established that at least one type of striatonigral neuron is of medium-size. This observation has been confirmed by other workers using the retrograde transport of horseradish peroxidase (HRP) (BUNNEY & AGHAJANIAN, 1976 ; SZABO, 1979) or Herpes simplex virus (BAK, MARHAM, COOK & STEVENS, 1978). However, since retrogradely-transported markers only label the somata, and sometimes the proximal dendrites of efferent neurons, information concerning the morphology of the efferent neuron is limited. The morphology and ultrastructural features of one type of striatonigral neuron have been described by SOMOGYI & SMITH (1979) using a combination of the retrograde transport of HRP and Golgi-staining in one animal. It was demonstrated that the mediumsize densely spinous neuron of the neostriatum can be retrogradely-Iabelled by injection of HRP in the sub2142 1. P. BOLAM er (11. stantia nigra. These observations have been confirmed (SOMOGYI, HODGSON & SMITH, 1979; SOMOGYI, BOLAM & SMITH, 1981(/) and are supported by the results of PRESTON, BISHOP & KiTAI (1980), who showed that medium-size densely spinous neurons, identified by intracellular injection of HRP, may occasionally be antidromically stimulated from the substantia nigra. However. these findings do not exclude the occurrence of a second type of striatonigral neuron. This is made likely by the presence of two putative transmitters, :'-aminobutyrate (G ABA) and substance p, in the striatonigral pathway (BROWNSTEIN, MROZ. TAPPAZ & LEEMAN, J 977: GALE, HONG & GUIDOTT!, 1977: KANAZAWA , EMSON & CUELLO, 1977: JESSELL, ErvIsoN , PAXINOS & CUELLO, 1978: STAINES. NAGY, VINCENT & FlBICiER. 1980) More direct evidence of a second type of striatal efferent neuron has come from retrograde tracing studies. In addition to medium-size striatal neurons labelled with Herpes virus following injection in the substantia nigra, BA K er ([/. (1978) also observed a small proportion of large labelled neurons. SZAI3() (1979) detected occasional large striatal neurons in the cat and monkey that were retrogradelylabelled with HR P following injection into the substantia nigra and in a report by GROFOVA (1979) some large striatal neurons. also in the C'It. were labelled following HR P injection into the retrorubral nucleus. This area has been suggested to be a dorsal extension of the zona compacta of the nigra (SZAl3(), 1979). We have looked for a second type of striatonigral neuron in rats that received injections of HRP in the substantia nigra. The ipsilateral neostriatum from each of these animals was processed to reveal HR P activity and was then impregnated by the Golgi method , sometimes followed by gold-toning. Many striatonigral neurons, that were labelled with HRP, were also Golgi-stained but these were all of the medium-size densely sp iny type and studies on these have been described elsewhere (BOLAM, SOMOGYI & SMITH, 1980: SOMOGYI er u/., 198 1 (/) In the same material. some larger striatal neurons were also retrogradely-Iabelled with HRP. We have now studied these neurons in the electron microscope and compared their cytological characteristics and afferent synaptic boutons with those of Golgi-impregnated neurons. EXPERIME TAL PROCEDURES All expel'iments were performed on female albino Wistar nits (150170g) using the stel'eotaxic a tlas of KC) NIG & KUPPEL (1963) The rats were anaesthetized with chloral hydrate (350 mg/kg i.p. in 0.9"" w/ v NaCI) and injected into the subst(lntia nigra with 6"" HRP conjugated with whealgerm agglutinin (WGA). prepared ~lccording to the method of GONATi\S. HARPER, MIZUTA'i1 & GO"i .,qAS (1979). The stereotaxic co-ordinates were: A. 2.2: L 6.2: V, 6.4 using an oblique approach at an angle of 45. The results of studies on HR P-Iabelled neurons were obtained from three rats. Rat I received 20 nl of the HRP-W G A conjugate, rat 2 35 nl and rat 3 30 n1. All injections were made using glass micro-pipettes (tip diameter 20-30 pm) and a controlled gas pressure system (SmIOGYI er 0/, 1979). Rat 1 and some of the animals from which Golgi -stained neurons were studied had received multiple electrolytic lesions in the cortex , as described previously (SOMOGYI et ai , 19810). Twenty hours following the injection of HR P the animals were re-anaesthetized and perfused through the heart with approximately 20 ml of 0.9 ~,o (w/ v) NaCI followed by approximately 200 ml of fixative consisting of 250;, glutaraldehyde, OS\, paraformaldehyde in 0.1 !vi phosphate buffer (pH 7.4). The area of HR P injection, i.e. the mesencephalon, was removed and washed in buffer followed by 15~/o sucrose in buffer. Forty pm cryostat sections were then taken and stained for HR P histochemistry using o-tolidine (dimethylbenzidine) (IS a substrate, or were Nissl stained. The neostriatum was then sliced in I mm thick blocks (necessary for the demonstration of HRP endproduct and Golgi impregnation in the same material), pl'ocessed to reveal retrogradely-transported HRP and subsequently Golgi-stained. sectioned at 80lim with a tissue chopper and the majority of the sections were then gold-toned . The combined HRP histochemistry Golgi-staining procedure has been described in detail elsewhere (SOMOGYI t.:I ([I .. 1979 : SOivlOGYI e{ al. . 1981a). The Golgi-sections were examined in the light-microscope ; cells of interest (i.e. HRP-Iabelled perikarya) were photographed, their position within the striatum noted and they were re-embedded for examination in the electron microscope. Golgi-stained and gold-toned neurons were also draWl) using a Leitz Camera Lucida and a X 100 oil immersion objective. Serial ultralhin sections (silver/grey) were mounted in single slot formvar-coated grids and examined using Cl Philips 201 C electl'on microscope with 20-· 30 lim objecti ve apertures. In order to improve contl'ast the Golgi sections were stained ell hloc with uranyl acetate cll1d the ultrathin sections with lead citrate (SOMOGYI, J 978) All the measurements given in the paper were made on Golgi section s and have not been corrected for shrinkage.