6. Whittaker, V.P., Michaelson, J.A., Kirkland, R.J.A.: Biochem. J. 90, 293 (1964) 7. Morgan, I.G., Vincedon, G., Gombos, G.: Biochim. Biophys. Acta 320, 671 (1973)
Retrograde Transport of Horseradish Peroxidase in Afferent and Efferent Neurons from Masseter Muscle in the Rat C. Hinrichsen* Nobel Institute for Neurophysiology, Karolinska Institutet, Stockholm, Sweden Electron-microscope [1] and microelectrode [2] studies indicate that a percentage of cells in the trigeminal mesencephalic nucleus are electronically coupled. It is important to identify whether coupled cells subserve the same or different masticatory muscles and/or periodontal ligament receptors shown to be represented in the nucleus [3, 4]. It has also been suggested that the contralateral masticatory musculature is represented in the nucleus [5]. Retrograde axonal transport of horseradish peroxidase (HRP) represents a precise physiologic method whereby neurons supplying a region can be labeled and identified. Uptake of HRP by first-order afferent fibers subserving encapsulated receptors, however, appears minimal in the adult, as the perineurium serves as a diffusion barrier to the protein [6]. To circumvent this problem to some extent in the study of afferent or efferent systems, HRP has been injected into the periphery of neonates before the perineurium becomes an effective barrier[7, 8] (Fig. 1A), applied directly to severed nerve trunks [9] and to exposed (and damaged) nerve endings of tooth pulp[10]. In the study of the central projections of Masseter muscle in the rat it has been found that incorporation of 1.6 mg% hyalnronidase (Sigma) [11]in an i.m. injection of 0.1 ml Ringer solution containing 2 mg HRP (Sigma Type VI) overcame
the problem of the perineural barrier in the adult. The injection by fine hypodermic needle did not cause macroscopic dissociation of the muscle but led to significant uptake of HRP by neurons of the trigeminal mesencephalic and motor nuclei (Fig. 1B and C). Similar injections of HRP without hyaluronidase were not successful in labeling neurons in the adult but did label neurons in the neonate (Fig. 1A). In the latter case, however, technical problems associated with size of the animal severely restrict the usefulness of the method. This study has shown that neurons labeled after intramuscular injection occur singly (varying from small to large cells) and in close apposition in clusters with either labeled or unlabeled cells (Fig. 1C). Clustering cells appears to involve cells from different peripheral fields. In no case after a unilateral injection has HRP been taken up by cells of the contralateral nucleus, nor was this shown in other experiments where HRP was injected stereotactically into the mesencephalic nucleus. Received March 19, 1975 1. Hinrichsen, C.F.L., Larramendi, L.M.H.: Brain Res. 7, 296 (1968) 2. Baker, R., Llinfis, R. : J. Physiol. 212, 45 (1971) 3. Corbin, K.B., Harrison, F. : J. Neurophysiol. 3, 423 (1940) 4. Jerge, C.R. : ibid. 26, 379 (1963) 5. Smith, R.D., Marcarian, H.Q., Niemer, W.T. : J. Comp. Neurol. 131, 79 (1967) 6. Kristensson, K., Olsson, Y.: Acta Neuropath. 17, 127 (1971) 7. Kristensson, K., Olsson, Y., Sj6strand, J.: Brain Res. 32, 399 (1971) 8. Gacek, R.R. : Exp. Neurol. 44, 381 (1974) 9. de Vito, J.L., Clausing, K.W., Smith, O.A. : Brain Res. 82, 269 (1974) 10. Furstman, L., Saporta, S., Kruger, L. : ibid. 84, 320 (1975) 11. Kirby, C.K., Eckenhoff, J.E., Looby, J.P.: Ann. N.Y. Acad. Sci. 52, 1166 (1950)
Erythrocyte Membrane Antigenicity and Intracellular Peroxidatic Reactions D.J. Giannitsis Abt. ftir Hfimostaseologie und Transfusionsmedizin der UniversitfitsklinikenHomburg/Saar, W.Germany
Fig. 1. (A) Photomicrograph of cells of the trigeminal mesencephalic nucleus in a 2-day-old rat 24 h after injection of HRP in the Masseter muscle. (B) Photomicrograph of trigeminal motor nucleus cell in adult rat 24 h following injection of HRP with hyaluronidase into Masseter muscle, (C) Photomicrograph of cells of the trigeminal mesencephalic nucleus in an adult rat 24 h following injection of HRP with hyaluronidase in Masseter muscle. Some cells in clusters show labelling with HRP (arrows) (bars 10 pm) * On leave of absence from the Department of Anatomy, University of Tasmania, Hobart, Tasmania, Australia. 492
It was suggested some time ago that cell-membrane receptors may play a role in the regulation of cell metabolism [1, 3]. The antigen antibody interactions may change the structure and functions of the membrane receptors [3, 4]. It is known that several immunoreactions are accompanied by changes in erythrocyte membrane-bound enzymes [4] and probably of the intracellular metabolism. The importance of the role of peroxidatic reactions to intact erythrocytes is demonstrated [4]. Normal red cells are rich in catalase (E.C. !.2.1.6; HzO2H202 oxidoreductase) [2]. We report here that interactions of the membrane-specific AB0-blood group receptors of normal human erythrocytes, with homologous as well as heterologous isoagglutinins, are accompanied by alterations of intracellular peroxidatic reactions. Human erythrocytes and isoagglutinins (sera; anti-A and antiB, free from irregular antibodies) were obtained from fresh ACD blood (plastic bags) of A 1, A2, B, O, and AB Rh-positive donors. The erythrocytes were separated from the blood plasma after centrifugation at 600 g for 5 min and were washed 6 times with 0.9% saline, after which the sediment of the last centrifugation was resuspended in Krebs-Ringer phosNaturwissenschaften 62 (1975) 9 by Springer-Verlag 1975
Retrograde Transport of Horseradish Peroxidase in Afferent and Efferent Neurons from Masseter Muscle in the Rat C. Hinrichsen* Nobel Institute for Neurophysiology, Karolinska Institutet, Stockholm, Sweden Electron-microscope [1] and microelectrode [2] studies indicate that a percentage of cells in the trigeminal mesencephalic nucleus are electronically coupled. It is important to identify whether coupled cells subserve the same or different masticatory muscles and/or periodontal ligament receptors shown to be represented in the nucleus [3, 4]. It has also been suggested that the contralateral masticatory musculature is represented in the nucleus [5]. Retrograde axonal transport of horseradish peroxidase (HRP) represents a precise physiologic method whereby neurons supplying a region can be labeled and identified. Uptake of HRP by first-order afferent fibers subserving encapsulated receptors, however, appears minimal in the adult, as the perineurium serves as a diffusion barrier to the protein [6]. To circumvent this problem to some extent in the study of afferent or efferent systems, HRP has been injected into the periphery of neonates before the perineurium becomes an effective barrier[7, 8] (Fig. 1A), applied directly to severed nerve trunks [9] and to exposed (and damaged) nerve endings of tooth pulp[10]. In the study of the central projections of Masseter muscle in the rat it has been found that incorporation of 1.6 mg% hyalnronidase (Sigma) [11]in an i.m. injection of 0.1 ml Ringer solution containing 2 mg HRP (Sigma Type VI) overcame
the problem of the perineural barrier in the adult. The injection by fine hypodermic needle did not cause macroscopic dissociation of the muscle but led to significant uptake of HRP by neurons of the trigeminal mesencephalic and motor nuclei (Fig. 1B and C). Similar injections of HRP without hyaluronidase were not successful in labeling neurons in the adult but did label neurons in the neonate (Fig. 1A). In the latter case, however, technical problems associated with size of the animal severely restrict the usefulness of the method. This study has shown that neurons labeled after intramuscular injection occur singly (varying from small to large cells) and in close apposition in clusters with either labeled or unlabeled cells (Fig. 1C). Clustering cells appears to involve cells from different peripheral fields. In no case after a unilateral injection has HRP been taken up by cells of the contralateral nucleus, nor was this shown in other experiments where HRP was injected stereotactically into the mesencephalic nucleus. Received March 19, 1975 1. Hinrichsen, C.F.L., Larramendi, L.M.H.: Brain Res. 7, 296 (1968) 2. Baker, R., Llinfis, R. : J. Physiol. 212, 45 (1971) 3. Corbin, K.B., Harrison, F. : J. Neurophysiol. 3, 423 (1940) 4. Jerge, C.R. : ibid. 26, 379 (1963) 5. Smith, R.D., Marcarian, H.Q., Niemer, W.T. : J. Comp. Neurol. 131, 79 (1967) 6. Kristensson, K., Olsson, Y.: Acta Neuropath. 17, 127 (1971) 7. Kristensson, K., Olsson, Y., Sj6strand, J.: Brain Res. 32, 399 (1971) 8. Gacek, R.R. : Exp. Neurol. 44, 381 (1974) 9. de Vito, J.L., Clausing, K.W., Smith, O.A. : Brain Res. 82, 269 (1974) 10. Furstman, L., Saporta, S., Kruger, L. : ibid. 84, 320 (1975) 11. Kirby, C.K., Eckenhoff, J.E., Looby, J.P.: Ann. N.Y. Acad. Sci. 52, 1166 (1950)
Erythrocyte Membrane Antigenicity and Intracellular Peroxidatic Reactions D.J. Giannitsis Abt. ftir Hfimostaseologie und Transfusionsmedizin der UniversitfitsklinikenHomburg/Saar, W.Germany
Fig. 1. (A) Photomicrograph of cells of the trigeminal mesencephalic nucleus in a 2-day-old rat 24 h after injection of HRP in the Masseter muscle. (B) Photomicrograph of trigeminal motor nucleus cell in adult rat 24 h following injection of HRP with hyaluronidase into Masseter muscle, (C) Photomicrograph of cells of the trigeminal mesencephalic nucleus in an adult rat 24 h following injection of HRP with hyaluronidase in Masseter muscle. Some cells in clusters show labelling with HRP (arrows) (bars 10 pm) * On leave of absence from the Department of Anatomy, University of Tasmania, Hobart, Tasmania, Australia. 492
It was suggested some time ago that cell-membrane receptors may play a role in the regulation of cell metabolism [1, 3]. The antigen antibody interactions may change the structure and functions of the membrane receptors [3, 4]. It is known that several immunoreactions are accompanied by changes in erythrocyte membrane-bound enzymes [4] and probably of the intracellular metabolism. The importance of the role of peroxidatic reactions to intact erythrocytes is demonstrated [4]. Normal red cells are rich in catalase (E.C. !.2.1.6; HzO2H202 oxidoreductase) [2]. We report here that interactions of the membrane-specific AB0-blood group receptors of normal human erythrocytes, with homologous as well as heterologous isoagglutinins, are accompanied by alterations of intracellular peroxidatic reactions. Human erythrocytes and isoagglutinins (sera; anti-A and antiB, free from irregular antibodies) were obtained from fresh ACD blood (plastic bags) of A 1, A2, B, O, and AB Rh-positive donors. The erythrocytes were separated from the blood plasma after centrifugation at 600 g for 5 min and were washed 6 times with 0.9% saline, after which the sediment of the last centrifugation was resuspended in Krebs-Ringer phosNaturwissenschaften 62 (1975) 9 by Springer-Verlag 1975
