Bra#l Research, 122 (1977) 229 242
229
(' Elsevier/North-Holland Biomedical Press, Amsterdam Printed in The Netherlands
H A B E N U L A R AND O T H E R MIDBRAIN RAPHE A F F E R E N T S DEMONSTRATED BY A M O D I F I E D R E T R O G R A D E T R A C I N G T E C H N I Q U E
G. K. A G H A J A N I A N and R E X Y. W A N G
Departments (~fPsychiatry and Pharmacology, Yale University School of Medicine, and the Connecticut Mental Health Center, New Haven, Conn. 06508 (U.S.A.) (Accepted June l lth, 1976)
SUMMARY
Afferents to the midbrain dorsal and median raphe nuclei in the rat were studied by means of the horseradish peroxidase (HRP) retrograde transport method. The H RP was given by means of a modified iontophoretic delivery technique. This technique permitted an efficient and localized deposition of a high concentration of HRP into the raphe nuclei. Afferents to the raphe as determined by this method could be categorized into 2 classes: those exclusively to the raphe and those also positive for adjacent reticular formation. The most striking afferent area to the raphe, both in terms of selectivity and density, was the lateral habenula. This result is in accord with previous studies using degeneration methods which indicate an habenular projection to the raphe area. There were afferents exclusively positive for the dorsal raphe nucleus emanating from the nucleus of the solitary tract. Most other raphe afferent areas were also positive for the reticular formation (e.g., prefrontal cortex, medial forebrain bundle, preoptic nuclei, and reticular formation). The existence of a major afferent system from the lateral habenula to the midbrain raphe is consistent with the concept of a "dorsal pathway" which might be responsible for relaying information from forebrain limbic structures to the "midbrain limbic areas".
INTRODUCTION
The brain stem raphe nuclei have been shown to contain most of the cells of origin of the serotonergic projections within the brain 11. The serotonergic system through its extensive efferents has been implicated in a great variety of physiological and behavioral functions (see ref. 9). However, relatively little is known about the afferent connections of the raphe nuclei. On the basis of lesion and degeneration studies there appear to be neuronal inputs to the raphe from prefrontal cortex5,'~6, 34, spinal cord~, cerebellum 5, caudate nucleus ~8, rostral basal forebrain 29, lateral hypo-
230 thalamuslV,a'~L and perhaps most strikingly, from the habenular nuclei z,:~:~ I here are also data from biochemical and histochemical studies that show a catccholamine input (either norepinephrine or epinephrine), particularly to the area i ihc dorsal raphe nucleus of the midbrain r',~:',~v,aS. Finally, there are data frcm~ the retrograde axonal transport method using horseradish peroxidase (H RP) ga,~a suggesting the occurrence of interconnections between raphe neurons ~. However. in the latter stud} and in our own preliminary experiments lunpublished) using a n3icrolite~ syringe delivery system for depositing H R P in the raphe nuclei, afferents from oti~er brain areas (even those described in the degeneration studies) were nol consistently observed. One reason for these apparent false negative results, a phenomenon which has been noted in other studies as well :~e. is that the raphe nuclei arc extremely small: even with the use of small volumes and line cannulae, hydraulic i~\iection,, tend t~, produce tissue damage and diffusion from the injection site. An alternative technique has been described for the delivery of H R P using electrophoresis through micro- or semi-micropipettes 15. Using a modification of this microelectrophoretic technique, we have been able to deposit extremely high concentrations of H R P reliably into discrete areas of the raphe nuclei with little tissue damage or diffusion. By this means, we have consistently been able to demonstrate raphe afferents emanating from many parts of the brain stem and tk~rebrain including most of the areas described in the degeneration studies. M ETHODS Twenty albino rats (Charles River Inc.), weighing 225-250 g, were used in these experiments. In preparation for the placement of H R P into the raphe and adjacent nuclei, animals were anesthetized with chloral hydrate (400 mg/kg) and mounted in a stereotaxic instrument. A burr hole was drilled with its center 0.5 1.0 mm anterior to lambda through which the micropipette ~as lowered. For the raphe placements the burr hole was in the midline and for placements in the reticular fl)rmation the hole was drilled 0.5-1.0 mm lateral to the midline.
Preparation of micropipelles In agreement with Graybiel and Dew)r 1:', it was found, in preliminary attempts, that the impedances of electrodes containing H R P solutions rose very rapidly when direct iontophoretic currents were used. However, the use of alternating currents 1:' was found to be inefficient presumably because the two active isozymes of Sigma type VI H R P are basic proteins ~ and would be ejected best by positive iontophoretic currents. It was found that a positive direct current could be used only if the pipettes were loaded with a few strands of fiber glass prior to pulling, as described by Tasaki et al. 3v. Micropipettes pulled in this manner were then broken back to a tip diameter of 25-50 #m under microscopic control using the microscope stage as a micromanipulator. The pipettes were then filled with an HRP solution (25 °,~i Sigma type VI in 0.01 M NaCI). The impedances of such electrodes (measured at 60 Hz through 0.9 Y/o NaCI) were usually between 5 and l0 M~2.
231
Microiontophoresis of HRP The pipettes were connected to a high-voltage constant current source (via a silver or platinum wire) prior to lowering into the brain. This was done to minimize extraneous manipulations which might result in lateral movements of the pipette; such movements cause tissue damage and excessive HRP diffusion. After rinsing the tip in saline, the pipette was carefully lowered into the raphe or adjacent regions. in our rats, a depth of 6.0 mm and 7.5 mm from skull surface was usually correct for dorsal (n -- 8) and median raphe (n = 5) placements, respectively. In 2 animals ejections were placed in both raphe nuclei and in 5 animals the placement was lateral to the raphe. The ejection of H R P was carried out at a current of 4 #A for 2-4 min. At these parameters a dense core of HRP reaction product having a diameter of approximately 0.25-0.5 mm was obtained. Histochemical reaction for HRP The procedure employed was a slight modification of the original technique described by Graham and Karnovsky 14. Twenty-eight to 48 h after the microiontophoretic ejection of HRP the animals were anesthetized and the brain fixed by intracardiac perfusion with a solution consisting of l ~ paraformaldehyde and 1 glutaraldehyde in 0.05 M phosphate buffer (pH 7.3, 22 °C). The brain was then removed and placed in 0.05 M Tris buffer (pH 7.6) containing 5 ~o sucrose. After storage overnight at 4 °C, frozen serial frontal sections (50 /~m) of the brain were cut. Each section was immediately transferred to one chamber of a multi-chambered tray. Each chamber contained 3 ml of a freshly filtered 1.4 m M solution of 3,3'diaminobenzidine (DAB) in 0.05 M Tris buffer (pH 7.6). The trays were incubated for 30 min at 22 °C. After cooling to room temperature, 0.4 ml of a 0.03 ~ solution of HzO2 was added to each chamber and the trays were then gently agitated for 1 h. Sections were mounted and examined in a light microscope using both brightand dark-field illumination. The latter mode of illumination is particularly effective in demonstrating the discrete granular HRP reaction product in afferent areas 24. RESULTS The microiontophoretic delivery method resulted in a nearly spherical core around which could be seen a less dense halo of reaction product. This is illustrated for a typical ejection into the midbrain dorsal raphe nucleus (Fig. 1). Note that the reaction at the center of the ejection site is so intense that the core zone (~0.5 ram) is virtually opaque. Also note that the zone of diffusion surrounding the core is confined to the dorsal raphe area and that there is little evidence of gross tissue damage as is seen after hydraulic injections. By dark-field examination, cells surrounding the dense core exhibited a mostly diffuse pattern of reaction product in which intracellular granules were poorly demarcated. This diffuse reaction product may represent local uptake of HRP by neuronal perikarya and dendrites 2s with an incomplete incorporation into cytoplasmic organelles 31. However, a similar diffuse pattern of HRP reaction product was found in raphe cells when the ejection site
232
•~
I~
Fig. I. Bright-field m i c r o g r a p h o f an H R P cjection into the midbrain dorsal i'api~e ~tlcicu~. 111
229
(' Elsevier/North-Holland Biomedical Press, Amsterdam Printed in The Netherlands
H A B E N U L A R AND O T H E R MIDBRAIN RAPHE A F F E R E N T S DEMONSTRATED BY A M O D I F I E D R E T R O G R A D E T R A C I N G T E C H N I Q U E
G. K. A G H A J A N I A N and R E X Y. W A N G
Departments (~fPsychiatry and Pharmacology, Yale University School of Medicine, and the Connecticut Mental Health Center, New Haven, Conn. 06508 (U.S.A.) (Accepted June l lth, 1976)
SUMMARY
Afferents to the midbrain dorsal and median raphe nuclei in the rat were studied by means of the horseradish peroxidase (HRP) retrograde transport method. The H RP was given by means of a modified iontophoretic delivery technique. This technique permitted an efficient and localized deposition of a high concentration of HRP into the raphe nuclei. Afferents to the raphe as determined by this method could be categorized into 2 classes: those exclusively to the raphe and those also positive for adjacent reticular formation. The most striking afferent area to the raphe, both in terms of selectivity and density, was the lateral habenula. This result is in accord with previous studies using degeneration methods which indicate an habenular projection to the raphe area. There were afferents exclusively positive for the dorsal raphe nucleus emanating from the nucleus of the solitary tract. Most other raphe afferent areas were also positive for the reticular formation (e.g., prefrontal cortex, medial forebrain bundle, preoptic nuclei, and reticular formation). The existence of a major afferent system from the lateral habenula to the midbrain raphe is consistent with the concept of a "dorsal pathway" which might be responsible for relaying information from forebrain limbic structures to the "midbrain limbic areas".
INTRODUCTION
The brain stem raphe nuclei have been shown to contain most of the cells of origin of the serotonergic projections within the brain 11. The serotonergic system through its extensive efferents has been implicated in a great variety of physiological and behavioral functions (see ref. 9). However, relatively little is known about the afferent connections of the raphe nuclei. On the basis of lesion and degeneration studies there appear to be neuronal inputs to the raphe from prefrontal cortex5,'~6, 34, spinal cord~, cerebellum 5, caudate nucleus ~8, rostral basal forebrain 29, lateral hypo-
230 thalamuslV,a'~L and perhaps most strikingly, from the habenular nuclei z,:~:~ I here are also data from biochemical and histochemical studies that show a catccholamine input (either norepinephrine or epinephrine), particularly to the area i ihc dorsal raphe nucleus of the midbrain r',~:',~v,aS. Finally, there are data frcm~ the retrograde axonal transport method using horseradish peroxidase (H RP) ga,~a suggesting the occurrence of interconnections between raphe neurons ~. However. in the latter stud} and in our own preliminary experiments lunpublished) using a n3icrolite~ syringe delivery system for depositing H R P in the raphe nuclei, afferents from oti~er brain areas (even those described in the degeneration studies) were nol consistently observed. One reason for these apparent false negative results, a phenomenon which has been noted in other studies as well :~e. is that the raphe nuclei arc extremely small: even with the use of small volumes and line cannulae, hydraulic i~\iection,, tend t~, produce tissue damage and diffusion from the injection site. An alternative technique has been described for the delivery of H R P using electrophoresis through micro- or semi-micropipettes 15. Using a modification of this microelectrophoretic technique, we have been able to deposit extremely high concentrations of H R P reliably into discrete areas of the raphe nuclei with little tissue damage or diffusion. By this means, we have consistently been able to demonstrate raphe afferents emanating from many parts of the brain stem and tk~rebrain including most of the areas described in the degeneration studies. M ETHODS Twenty albino rats (Charles River Inc.), weighing 225-250 g, were used in these experiments. In preparation for the placement of H R P into the raphe and adjacent nuclei, animals were anesthetized with chloral hydrate (400 mg/kg) and mounted in a stereotaxic instrument. A burr hole was drilled with its center 0.5 1.0 mm anterior to lambda through which the micropipette ~as lowered. For the raphe placements the burr hole was in the midline and for placements in the reticular fl)rmation the hole was drilled 0.5-1.0 mm lateral to the midline.
Preparation of micropipelles In agreement with Graybiel and Dew)r 1:', it was found, in preliminary attempts, that the impedances of electrodes containing H R P solutions rose very rapidly when direct iontophoretic currents were used. However, the use of alternating currents 1:' was found to be inefficient presumably because the two active isozymes of Sigma type VI H R P are basic proteins ~ and would be ejected best by positive iontophoretic currents. It was found that a positive direct current could be used only if the pipettes were loaded with a few strands of fiber glass prior to pulling, as described by Tasaki et al. 3v. Micropipettes pulled in this manner were then broken back to a tip diameter of 25-50 #m under microscopic control using the microscope stage as a micromanipulator. The pipettes were then filled with an HRP solution (25 °,~i Sigma type VI in 0.01 M NaCI). The impedances of such electrodes (measured at 60 Hz through 0.9 Y/o NaCI) were usually between 5 and l0 M~2.
231
Microiontophoresis of HRP The pipettes were connected to a high-voltage constant current source (via a silver or platinum wire) prior to lowering into the brain. This was done to minimize extraneous manipulations which might result in lateral movements of the pipette; such movements cause tissue damage and excessive HRP diffusion. After rinsing the tip in saline, the pipette was carefully lowered into the raphe or adjacent regions. in our rats, a depth of 6.0 mm and 7.5 mm from skull surface was usually correct for dorsal (n -- 8) and median raphe (n = 5) placements, respectively. In 2 animals ejections were placed in both raphe nuclei and in 5 animals the placement was lateral to the raphe. The ejection of H R P was carried out at a current of 4 #A for 2-4 min. At these parameters a dense core of HRP reaction product having a diameter of approximately 0.25-0.5 mm was obtained. Histochemical reaction for HRP The procedure employed was a slight modification of the original technique described by Graham and Karnovsky 14. Twenty-eight to 48 h after the microiontophoretic ejection of HRP the animals were anesthetized and the brain fixed by intracardiac perfusion with a solution consisting of l ~ paraformaldehyde and 1 glutaraldehyde in 0.05 M phosphate buffer (pH 7.3, 22 °C). The brain was then removed and placed in 0.05 M Tris buffer (pH 7.6) containing 5 ~o sucrose. After storage overnight at 4 °C, frozen serial frontal sections (50 /~m) of the brain were cut. Each section was immediately transferred to one chamber of a multi-chambered tray. Each chamber contained 3 ml of a freshly filtered 1.4 m M solution of 3,3'diaminobenzidine (DAB) in 0.05 M Tris buffer (pH 7.6). The trays were incubated for 30 min at 22 °C. After cooling to room temperature, 0.4 ml of a 0.03 ~ solution of HzO2 was added to each chamber and the trays were then gently agitated for 1 h. Sections were mounted and examined in a light microscope using both brightand dark-field illumination. The latter mode of illumination is particularly effective in demonstrating the discrete granular HRP reaction product in afferent areas 24. RESULTS The microiontophoretic delivery method resulted in a nearly spherical core around which could be seen a less dense halo of reaction product. This is illustrated for a typical ejection into the midbrain dorsal raphe nucleus (Fig. 1). Note that the reaction at the center of the ejection site is so intense that the core zone (~0.5 ram) is virtually opaque. Also note that the zone of diffusion surrounding the core is confined to the dorsal raphe area and that there is little evidence of gross tissue damage as is seen after hydraulic injections. By dark-field examination, cells surrounding the dense core exhibited a mostly diffuse pattern of reaction product in which intracellular granules were poorly demarcated. This diffuse reaction product may represent local uptake of HRP by neuronal perikarya and dendrites 2s with an incomplete incorporation into cytoplasmic organelles 31. However, a similar diffuse pattern of HRP reaction product was found in raphe cells when the ejection site
232
•~
I~
Fig. I. Bright-field m i c r o g r a p h o f an H R P cjection into the midbrain dorsal i'api~e ~tlcicu~. 111
