Brain Research, 88 (1975) 403~14
403
(~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Research Reports
R E T R O G R A D E T R A N S P O R T OF H O R S E R A D I S H P E R O X I D A S E IN T H E M A G N O C E L L U L A R N E U R O S E C R E T O R Y SYSTEM OF T H E R A T
D. A. SHERLOCK, P. M. FIELD AYD G. RAISMAN
Laboratory o[ Neurobiology, National Institute ]or Medical Research, Mill Hill, London NW7 IAA (Great Britain) (Accepted December 7th, 1974)
SUMMARY
Horseradish peroxidase (HRP) was injected into the pituitary in adult rats. After 2-3 days, the neurones of the supraoptic nuclei, the magnocellular parts of the paraventricular nuclei, and the various accessory neurosecretory hypothalamic nuclei showed accumulation of HRP. The H R P reaction product consisted of fine, discrete cytoplasmic granules, and in electron micrographs it was seen to be located in the lysosome-like dense bodies of 0.4-0.6 # m diameter which are normally present in the cytoplasm of the neurosecretory neurones. Very little reaction product was found in the neurosecretory axons. Reaction product was also found in the hypothalamic arcuate nuclei. This was the result of an endogenous peroxidase-like activity, since it occurred in control animals which had not received HRP. This endogenous reaction product is nonneuronal. Morphologically, it takes the form of distinctive clusters of coarse granules which are seen in electron micrographs to be characteristic angular bodies of 0.7-1.0 /~m diameter located in the cytoplasm of astrocytes or their processes.
INTRODUCTION
The retrograde axonal transport of horseradish peroxidase (HRP) which was originally demonstrated in peripheral motoneurones 15, and later in the central nervous system 16, has aroused a great deal of interest among neuroanatomists. This application of H R P is useful as a tracer technique for identifying the cells of origin of fibre projections to specific regions17, 24. Moreover, as a direct means of demonstrating how material taken up in nerve terminals can be transported to the neuronal perikaryon, it has potential importance for studying the ways by which factors in the distri-
404 bution field of neurones might regulate the growth and maintenance ~H" nerve ~cils and their axonal arborizations ~;~. In the present study we have examined the retrograde transport of HRP in the magnocellular neurosecretory system of the rat hypothalamus. The large cells tH the supraoptic and magnocellular parts of the paraventricular nuclei, together x~itla scattered cells in the accessory neurosecretory nuclei 2(~, have axons which pass through the internal zone of the median eminence and pituitary stalk to terminate on [he fenestrated capillaries of the posterior pituitary. These cells have a characteristic morphology, and synthesize neurosecretory material which can be identified hi~t,)chemically and ultrastructurally, and which is carried orthogradely down the ax~)ns to be released into the capillaries ~. After axotomy the cells show a marked retrograde reaction; a proportion of the cells ultimately degenerate 27,3° and tile remainder regenerate an extensive terminal plexus around a mass of newly proliferated fenestrated capillaries referred to as a miniature neural lobe "°,2~,2s,33. It has already becn demonstrated ultrastructurally that the neurosecretory axon terminals in the ~at posterior pituitary take up extracellular H R P into coated and smooth microvesicles, and this has been used as evidence for the recapture of excess membrane let1 as a result of an exocytotic secretion process ~:~. MATERIAL AND METHODS
Fifteen young female albino rats of the Wistar strain, weighing around 200 g, were used for injection of H R P into the pituitary. In addition a series of unoperated rats and also rats with injections of H R P into the mammillary nuclei were available as controls. Under anaesthesia with Avertin (Winthrop, 182 mg/kg, i.p.) a stereotaxically guided glass micropipette (tip diameter 60-70 /~m) was inserted into the pituitary through a small drill hole in the midline 2.5 m m caudal to the bregma. Using a mechanical microdrive pump, acting on a paraffin-filled 1 /A Hamilton syringe connected by a short length of polythene tubing to the micropipette, between 0.6 and 0.9 ,ul of a 50-60 ~o aqueous solution of H R P (Sigma Type VI) was injected from the pipette over a period of 13-20 rain. The rats were allowed to survive for 2-3 days after operation, during which time they were dehydrated to ensure a high level of activity of the osmosensitive neurosecretory neurones (and hence possibly an increased rate of uptake of H RpI0,z3). Under anaesthesia the rats were perfused transcardially with 200 nil of saline at 4 °C followed by I litre of a mixture of 1 ~ formaldehyde and 1 ')/, glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 and 4 °C. Tile brains were removed, placed in fresh fixative on a shaker for a further 4-6 h, and then stored overnight in phosphate buffer with 5"/ ~o sucrose and 1 ~o dimethylsulphoxide. Frozen sections were cut at 40-50 # m in the coronal or sagittal planes and processed to demonstrate the presence of H R P (30-60 rain incubation in diaminobenzidine and hydrogen peroxide; for details see refs. 8, 17, 24). Sections, either unstained or lightly counterstained with cresyl violet or thionin, were examined in light field, dark field, or with interference contrast optics (Leitz, Wetzlar). For electron microscopy, sections were cut coronally at 100 # m either on the freezing mierotome, or on the Vibratome (Oxford Instruments, Calif.) to avoid freeze damage to the sec-
405 tions. Alter pr,~ccssing to show the presence of HRP, the sections were left in buffer or fixative overnight. Under a dissecting microscope the parts of the sections containing the supraoptic uuclei, the paraventricular nuclei, the arcuate nuclei and the median eminence were cat ~Jut with a razor blade and postfixed in 2 ~ osmium tetroxide in phosphate buffer at pH 7.3, dehydrated and embedded flat in Epon. R ESU LTS
Injection sites In 8 experiments peroxidase reaction product was diffused throughout the entire pituitar> T:is ~vas due partly to the injected (exogenous) HRP, and partly to the peroxida:< reaction of extravasated blood associated with direct tissue damage. All injection, s
403
(~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Research Reports
R E T R O G R A D E T R A N S P O R T OF H O R S E R A D I S H P E R O X I D A S E IN T H E M A G N O C E L L U L A R N E U R O S E C R E T O R Y SYSTEM OF T H E R A T
D. A. SHERLOCK, P. M. FIELD AYD G. RAISMAN
Laboratory o[ Neurobiology, National Institute ]or Medical Research, Mill Hill, London NW7 IAA (Great Britain) (Accepted December 7th, 1974)
SUMMARY
Horseradish peroxidase (HRP) was injected into the pituitary in adult rats. After 2-3 days, the neurones of the supraoptic nuclei, the magnocellular parts of the paraventricular nuclei, and the various accessory neurosecretory hypothalamic nuclei showed accumulation of HRP. The H R P reaction product consisted of fine, discrete cytoplasmic granules, and in electron micrographs it was seen to be located in the lysosome-like dense bodies of 0.4-0.6 # m diameter which are normally present in the cytoplasm of the neurosecretory neurones. Very little reaction product was found in the neurosecretory axons. Reaction product was also found in the hypothalamic arcuate nuclei. This was the result of an endogenous peroxidase-like activity, since it occurred in control animals which had not received HRP. This endogenous reaction product is nonneuronal. Morphologically, it takes the form of distinctive clusters of coarse granules which are seen in electron micrographs to be characteristic angular bodies of 0.7-1.0 /~m diameter located in the cytoplasm of astrocytes or their processes.
INTRODUCTION
The retrograde axonal transport of horseradish peroxidase (HRP) which was originally demonstrated in peripheral motoneurones 15, and later in the central nervous system 16, has aroused a great deal of interest among neuroanatomists. This application of H R P is useful as a tracer technique for identifying the cells of origin of fibre projections to specific regions17, 24. Moreover, as a direct means of demonstrating how material taken up in nerve terminals can be transported to the neuronal perikaryon, it has potential importance for studying the ways by which factors in the distri-
404 bution field of neurones might regulate the growth and maintenance ~H" nerve ~cils and their axonal arborizations ~;~. In the present study we have examined the retrograde transport of HRP in the magnocellular neurosecretory system of the rat hypothalamus. The large cells tH the supraoptic and magnocellular parts of the paraventricular nuclei, together x~itla scattered cells in the accessory neurosecretory nuclei 2(~, have axons which pass through the internal zone of the median eminence and pituitary stalk to terminate on [he fenestrated capillaries of the posterior pituitary. These cells have a characteristic morphology, and synthesize neurosecretory material which can be identified hi~t,)chemically and ultrastructurally, and which is carried orthogradely down the ax~)ns to be released into the capillaries ~. After axotomy the cells show a marked retrograde reaction; a proportion of the cells ultimately degenerate 27,3° and tile remainder regenerate an extensive terminal plexus around a mass of newly proliferated fenestrated capillaries referred to as a miniature neural lobe "°,2~,2s,33. It has already becn demonstrated ultrastructurally that the neurosecretory axon terminals in the ~at posterior pituitary take up extracellular H R P into coated and smooth microvesicles, and this has been used as evidence for the recapture of excess membrane let1 as a result of an exocytotic secretion process ~:~. MATERIAL AND METHODS
Fifteen young female albino rats of the Wistar strain, weighing around 200 g, were used for injection of H R P into the pituitary. In addition a series of unoperated rats and also rats with injections of H R P into the mammillary nuclei were available as controls. Under anaesthesia with Avertin (Winthrop, 182 mg/kg, i.p.) a stereotaxically guided glass micropipette (tip diameter 60-70 /~m) was inserted into the pituitary through a small drill hole in the midline 2.5 m m caudal to the bregma. Using a mechanical microdrive pump, acting on a paraffin-filled 1 /A Hamilton syringe connected by a short length of polythene tubing to the micropipette, between 0.6 and 0.9 ,ul of a 50-60 ~o aqueous solution of H R P (Sigma Type VI) was injected from the pipette over a period of 13-20 rain. The rats were allowed to survive for 2-3 days after operation, during which time they were dehydrated to ensure a high level of activity of the osmosensitive neurosecretory neurones (and hence possibly an increased rate of uptake of H RpI0,z3). Under anaesthesia the rats were perfused transcardially with 200 nil of saline at 4 °C followed by I litre of a mixture of 1 ~ formaldehyde and 1 ')/, glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 and 4 °C. Tile brains were removed, placed in fresh fixative on a shaker for a further 4-6 h, and then stored overnight in phosphate buffer with 5"/ ~o sucrose and 1 ~o dimethylsulphoxide. Frozen sections were cut at 40-50 # m in the coronal or sagittal planes and processed to demonstrate the presence of H R P (30-60 rain incubation in diaminobenzidine and hydrogen peroxide; for details see refs. 8, 17, 24). Sections, either unstained or lightly counterstained with cresyl violet or thionin, were examined in light field, dark field, or with interference contrast optics (Leitz, Wetzlar). For electron microscopy, sections were cut coronally at 100 # m either on the freezing mierotome, or on the Vibratome (Oxford Instruments, Calif.) to avoid freeze damage to the sec-
405 tions. Alter pr,~ccssing to show the presence of HRP, the sections were left in buffer or fixative overnight. Under a dissecting microscope the parts of the sections containing the supraoptic uuclei, the paraventricular nuclei, the arcuate nuclei and the median eminence were cat ~Jut with a razor blade and postfixed in 2 ~ osmium tetroxide in phosphate buffer at pH 7.3, dehydrated and embedded flat in Epon. R ESU LTS
Injection sites In 8 experiments peroxidase reaction product was diffused throughout the entire pituitar> T:is ~vas due partly to the injected (exogenous) HRP, and partly to the peroxida:< reaction of extravasated blood associated with direct tissue damage. All injection, s
