Brain Research, 587 (19{)2) 343-347 © 1992 Elsevier Science Publishers B,V. All rights reserved I)006-8993/92/$05,00
343
BRES 25296
Cholinergic denervation alters [3H]phorbol-12,13-dibutyrate binding to rat hippocampal membranes V i s w a p r a b h u Ayyagari ~ and Lindy E. H a r r e U
b
"Behavioral Nem'oscience Program, ,.b Departments of Neurology and Psychology b VeteransAdministration and ,,,h Unit'ersity ojfAlabama at Birminghmn, Birmingham, AL 35294-0017, USA
Accepted 5 May 1992
Key words: PKC, phorbol; Hippocampus; Neuronal plasticity; Signal transduction; Cholinergic: Protein kinase C; Isozyme; Rat; Receptor binding; Sprouting; Sympathetic neuron
Cholinergic denervation of the rat hippocampus caused by electrolytic lesions of the medial septum (MS) results in a time-bound ingrowth of peripheral sympathetic noradrenergic fibers from the superior cervical ganglion to the dentate gyrus and CA3 region of the Uppocampus. To determine the functional significance of hippocampal sympathetic ingrowth (HSI), ['~H]phorbol-12,13-dibutyrate (PDBu) binding was assessed 4 weeks after MS lesions, in control animals, affinity for [~H]PDBu binding was found to be greater in the dorsal compared to ventral hippocampus, while the number of binding sites (Bin, x) was similar between regions. Regardless of the presence of HSI, MS lesions resulted in increased affinity in the dorsal hippocampus, while the Bm,~xwas found to 'normalize' in the ventral hippocampus by HSI. These results suggest that HSI is functional and can alter important cellular events,
The hippocampal formation is innervated by cholinergic and non-cholinergic neurons whose cell bodies reside in the septum ~,2,1,2x, Electrolytic lesions of the medial septum result not only in cholinergie denervation of the hippocampus, but also elicit an interesting example of functional neuronal plasticity. Following such cholinergic denervation, sympathetic noradrenergic fibers that are normally restricted to the parahippocampal blood vessels, invade the dentate gyrus and the CA3 region of the hippocampal formation J~. Hippocampal sympathetic ingrowth (HSI) is a functional neuronal reorgan'ization that has been found to affect a variety of both learned and non-learned behaviors 2..~.t2-t5. It is believed that behavioral changes occur as a result of alterations in cellular signal transduetion in the neuron. Recently, several signal transduction processes have been identified in the rat brain t l,2o.2.~.2s. The polyphosphoinositide second messenger system appears to be the major signal transduction mechanism associated with the muscarinie cholinergic receptor e.~. It was demonstrated more than thirty years ago, that
acetylcholine, via muscarinic receptor activation, promotes the turnover of phosphatidyl inositol ". This original finding has been substantiated and expanded by a number of more recent studies ~().~.2.~Thus, agonist occupation of the muscarinic acetylcholinc receptor (mAChR) stimulates the hydrolysis of the cell memI~rane-resident phospholipid, phosphatidyl inositol,4,5-bisphosphate (Ptdins-4,5-P.,) to yield two second messengers: sn.l,2-diacylglycerol (DAG) and inositol- 1,4,5-trisphosphate (! ns- 1,4,5-P.~). The water-soluble lns-l,4,5-P.~ diffuses into the cytosol and releases Ca 2+ from intracellular stores through binding to receptors on the endoplasmic retieulum, whereas the neutral DAG remains within the plane of the membrane, activating the enzyme protein kinase C (PKC). The accumulation of inositol-l-phosphate (lnsiP), measured in the presence of lithium, reflects the involvement of the Ins-l,4,5-P 3 branch of the Ptdlns-4,5P2 hydrolysis products. Previously, we have reported that the presence of HSI significantly altered the accumulation of inslP when the turnover of polyphosphoinositides was stimulated by carbachol but not NE 7.12.
Correspondence: V. Ayyagari, Departments of Neurology and Psychology, Room SC 910, University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA. Fax: (I) (2()5) 934-3709.
This effect was dose-dependent, followed the time course of ingrowth and was blocked by atropine, suggesting mediation through muscarinic cholinergic receptors. PKC is a closely related family of phospholipid-dependent diacylglycerol-activated serine/threonine protein kinases that play a fundamental role in cellular signal transduetion .,5. Activation of PKC has been linked to the regulation of cell surface receptors, ion channels, neurotransmittcr secretion, gene expression and neuronal plasticity 's. The molecular biology of PKC is presently under extensive investigation ,.,x. Of the several known isoforms of PKC, the activity of the isozymes expressed by the PKC a,/~!,/~!1, and the F genes are known to be dependent on the intracellular levels of Ca :+ (reL 17), the concentration of which is altered by the binding of lns-l,4,5-P~. Phorbol esters, which bind to two domains on the PKC molecule r,. mimic the action of DAG. Thus, these substances induce the translocation of cytosolic PKC to the cell membrane and activate PKC in the presence of calcium and phospholipids "~. Binding of t he phorbol ester [ '~H ]phorbol 12,13-dibutyrate (PD Bu) has previously been utilized as a means to measure PKC 4..~..,,J. These studies have demonstrated PDBu binding sites in the hippocampus as well as throughout the rat br:dn "~. Since we have previously demonstrated an effect of HSI on InslP accumulation '", we sought to determine whether this alteration in the polyphophoinositide second messenger system produces changes in subsequent cellular events by assessing the efl'~ct of HSI on ['~H]PDBu binding to rat hippocampal membranes. This work has appeared earlier as an abstract ~, Male Sprague-Dawley rats (initial body weight 150200 g)were housed in a temperature and light-con. trolled (12L: 12D)colony. One week after adaptation to the colony, animals were rm,domly assigned to one of three surgical procedures: CON (sham lesion of the medial septum and sham ganglionectomy [Gx]); MS/HS! (MS lesion + sham Gx); MS/Gx (MS lesion + Gx). All surgery was performed under kctamine (! l0 mg/kg; i.p.) and xylazene (13 mg/kg; i.p,) anesthesia with the neurosurgical and general surgical procedures performed during one anesthetic administration, Medial septal lesions (nose bar -2,5, coordinates from bregma, AP +0,8, ML 0, DV -5.9)were produced by passing direct current (3 mA for 15 s) through a bipolar, teflon-coated stainless steel electrode (tip diameter ! ram). Sham neurosurgical procedures were performed in the same manner except that no current was passed. The superior cervical ganglion (SCG) was removed
bilaterally via a ventral midline neck incision. Blunt dissection was performed to expose the bifurcation of the common carotid, the ganglia then visualized, and subsequently removed. Sham surgeries were performed in a similar manner except that the ganglia were left intact. Initial removal was assessed by examination for a Horner's syndrome (ptosis and myosis). Four to six weeks after surgery, animals were decapitated and brains removed within 60 s. A septal block was taken for verification of lesion placement and to assess the presence or absence of sympathetic fibers. The two hippocampii were dissected into ventral (VH) and dorsal (DH) regions and the corresponding regions combined for binding studies. Each animal was assayed separately. [3H]PDBu binding was performed according to published methods 4 with minor modifications. Briefly, tissue was homogenized using a Teflon and glass homogenizer, with 20 volumes of Tris buffer (Tris 50 raM, pH 7.7, containing 100 mM NaCi and 1 mM CaCI,) and centrifuged at 48,000 x g for 15 min at 0°C. The pellet (membrane fraction) was re-suspended in a Polytron at 50% pulse setting for 12 min. The binding assay was carried out for 150 rain in a final volume of ! ml of Tris buffer containing 25/~1 of the membrane fraction (corresponding to 100-125/zg of protein) in the presence of phorbol-12,13-dibutyrate [20-'~H(N)] lAmersham, 18.4 Ci/mmol). Binding was terminated by rapid filtration through Whatman GF/C filters presoaked for 15 min in 0.5% polyethylcnimine. Filters were washed 3 times with 3 ml Tris 50 mM, pH 7.'/, and the radioactivity determined by liquid scintillation counting using Budget Solve (RPI), Specific binding was defined as the difference between total binding and that in presence of unlabelled 10 /zM PDBu dissolved in DMSO. Protein concentration was determined by the method of Lowry ct al. ,2. Coronal sections (20/zm) were taken in duplicates at 100 #m intervals throughout the septum on an AO cryostat and stained for Cresyl violet and acetylcholine,,,terase :4. A third series of sections through the septum (every 3(X) /~m) was processed for norepinephrine histofluorescence ~. All data were analyzed using the appropriate model analysis of variance (unweighted means ANOVA:SAS) followed by Duncan's multiple range test for post-hoe testing, When appropriate, Least Squares Means was employed for comparisons between groups. Six rats were excluded from the study because of improperly placed lesions or death. Data from the remaining 14 animals (CON: n = 5; MS/HSI: n = 5; MS/Gx: n = 4) were used for further analysis. Histological examination of the lesioned animals revealed
345 MS lesions that were typical of those produced in our laboratory 2. Thus, in the MS/HSI and MS/Gx groups there was total destruction of the medial septal region throughout the entire anterior-posterior extent. Destruction of the ventral limb of the diagonal band of Broca was also found in all lesioned animals, with variable destruction of the horizontal limb. In the CON group, limited gliosis was observed along the electrode track, but damage to the cholinergic neurons was absent. Peripheral NE fibers were observed on the cerebral arteries and choroid plexus in both the CON and MS/HSI groups, while these fibers were absent in the MS/Gx group. in the CON group, the binding affinity (K d) of [3HI PDBu was found to vary by hippocampal region (Table I). Thus, the Ko was significantly greater in the VH compared to DH [F(~.m= 21.70, P < 0.01)]. Medial septal lesions, regardless of the presence or absence of HSI, resulted in an increased K d in the DH compared to the DH of controls (P < 0.05; Duncan's). No differences were observed in the affinity of [3H]PDBu binding in the ventral hippocampus when the MS lesioned groups were compared to each other or to controls (Fig. IA). in controls, the number of phorbol ester binding sites (Bmax) in the DH and the VH were similar (Table !). In the dorsal hippocampus, Bin,~ was similar among all experimental groups, suggesting no effect of MS lesions or HS! on the number of binding sites. However, in the VH, B,,,,x was found to be significantly greater in the MS/Gx group when compared to both the MS/HSI and the CON groups, which did not differ from each other [F
343
BRES 25296
Cholinergic denervation alters [3H]phorbol-12,13-dibutyrate binding to rat hippocampal membranes V i s w a p r a b h u Ayyagari ~ and Lindy E. H a r r e U
b
"Behavioral Nem'oscience Program, ,.b Departments of Neurology and Psychology b VeteransAdministration and ,,,h Unit'ersity ojfAlabama at Birminghmn, Birmingham, AL 35294-0017, USA
Accepted 5 May 1992
Key words: PKC, phorbol; Hippocampus; Neuronal plasticity; Signal transduction; Cholinergic: Protein kinase C; Isozyme; Rat; Receptor binding; Sprouting; Sympathetic neuron
Cholinergic denervation of the rat hippocampus caused by electrolytic lesions of the medial septum (MS) results in a time-bound ingrowth of peripheral sympathetic noradrenergic fibers from the superior cervical ganglion to the dentate gyrus and CA3 region of the Uppocampus. To determine the functional significance of hippocampal sympathetic ingrowth (HSI), ['~H]phorbol-12,13-dibutyrate (PDBu) binding was assessed 4 weeks after MS lesions, in control animals, affinity for [~H]PDBu binding was found to be greater in the dorsal compared to ventral hippocampus, while the number of binding sites (Bin, x) was similar between regions. Regardless of the presence of HSI, MS lesions resulted in increased affinity in the dorsal hippocampus, while the Bm,~xwas found to 'normalize' in the ventral hippocampus by HSI. These results suggest that HSI is functional and can alter important cellular events,
The hippocampal formation is innervated by cholinergic and non-cholinergic neurons whose cell bodies reside in the septum ~,2,1,2x, Electrolytic lesions of the medial septum result not only in cholinergie denervation of the hippocampus, but also elicit an interesting example of functional neuronal plasticity. Following such cholinergic denervation, sympathetic noradrenergic fibers that are normally restricted to the parahippocampal blood vessels, invade the dentate gyrus and the CA3 region of the hippocampal formation J~. Hippocampal sympathetic ingrowth (HSI) is a functional neuronal reorgan'ization that has been found to affect a variety of both learned and non-learned behaviors 2..~.t2-t5. It is believed that behavioral changes occur as a result of alterations in cellular signal transduetion in the neuron. Recently, several signal transduction processes have been identified in the rat brain t l,2o.2.~.2s. The polyphosphoinositide second messenger system appears to be the major signal transduction mechanism associated with the muscarinie cholinergic receptor e.~. It was demonstrated more than thirty years ago, that
acetylcholine, via muscarinic receptor activation, promotes the turnover of phosphatidyl inositol ". This original finding has been substantiated and expanded by a number of more recent studies ~().~.2.~Thus, agonist occupation of the muscarinic acetylcholinc receptor (mAChR) stimulates the hydrolysis of the cell memI~rane-resident phospholipid, phosphatidyl inositol,4,5-bisphosphate (Ptdins-4,5-P.,) to yield two second messengers: sn.l,2-diacylglycerol (DAG) and inositol- 1,4,5-trisphosphate (! ns- 1,4,5-P.~). The water-soluble lns-l,4,5-P.~ diffuses into the cytosol and releases Ca 2+ from intracellular stores through binding to receptors on the endoplasmic retieulum, whereas the neutral DAG remains within the plane of the membrane, activating the enzyme protein kinase C (PKC). The accumulation of inositol-l-phosphate (lnsiP), measured in the presence of lithium, reflects the involvement of the Ins-l,4,5-P 3 branch of the Ptdlns-4,5P2 hydrolysis products. Previously, we have reported that the presence of HSI significantly altered the accumulation of inslP when the turnover of polyphosphoinositides was stimulated by carbachol but not NE 7.12.
Correspondence: V. Ayyagari, Departments of Neurology and Psychology, Room SC 910, University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA. Fax: (I) (2()5) 934-3709.
This effect was dose-dependent, followed the time course of ingrowth and was blocked by atropine, suggesting mediation through muscarinic cholinergic receptors. PKC is a closely related family of phospholipid-dependent diacylglycerol-activated serine/threonine protein kinases that play a fundamental role in cellular signal transduetion .,5. Activation of PKC has been linked to the regulation of cell surface receptors, ion channels, neurotransmittcr secretion, gene expression and neuronal plasticity 's. The molecular biology of PKC is presently under extensive investigation ,.,x. Of the several known isoforms of PKC, the activity of the isozymes expressed by the PKC a,/~!,/~!1, and the F genes are known to be dependent on the intracellular levels of Ca :+ (reL 17), the concentration of which is altered by the binding of lns-l,4,5-P~. Phorbol esters, which bind to two domains on the PKC molecule r,. mimic the action of DAG. Thus, these substances induce the translocation of cytosolic PKC to the cell membrane and activate PKC in the presence of calcium and phospholipids "~. Binding of t he phorbol ester [ '~H ]phorbol 12,13-dibutyrate (PD Bu) has previously been utilized as a means to measure PKC 4..~..,,J. These studies have demonstrated PDBu binding sites in the hippocampus as well as throughout the rat br:dn "~. Since we have previously demonstrated an effect of HSI on InslP accumulation '", we sought to determine whether this alteration in the polyphophoinositide second messenger system produces changes in subsequent cellular events by assessing the efl'~ct of HSI on ['~H]PDBu binding to rat hippocampal membranes. This work has appeared earlier as an abstract ~, Male Sprague-Dawley rats (initial body weight 150200 g)were housed in a temperature and light-con. trolled (12L: 12D)colony. One week after adaptation to the colony, animals were rm,domly assigned to one of three surgical procedures: CON (sham lesion of the medial septum and sham ganglionectomy [Gx]); MS/HS! (MS lesion + sham Gx); MS/Gx (MS lesion + Gx). All surgery was performed under kctamine (! l0 mg/kg; i.p.) and xylazene (13 mg/kg; i.p,) anesthesia with the neurosurgical and general surgical procedures performed during one anesthetic administration, Medial septal lesions (nose bar -2,5, coordinates from bregma, AP +0,8, ML 0, DV -5.9)were produced by passing direct current (3 mA for 15 s) through a bipolar, teflon-coated stainless steel electrode (tip diameter ! ram). Sham neurosurgical procedures were performed in the same manner except that no current was passed. The superior cervical ganglion (SCG) was removed
bilaterally via a ventral midline neck incision. Blunt dissection was performed to expose the bifurcation of the common carotid, the ganglia then visualized, and subsequently removed. Sham surgeries were performed in a similar manner except that the ganglia were left intact. Initial removal was assessed by examination for a Horner's syndrome (ptosis and myosis). Four to six weeks after surgery, animals were decapitated and brains removed within 60 s. A septal block was taken for verification of lesion placement and to assess the presence or absence of sympathetic fibers. The two hippocampii were dissected into ventral (VH) and dorsal (DH) regions and the corresponding regions combined for binding studies. Each animal was assayed separately. [3H]PDBu binding was performed according to published methods 4 with minor modifications. Briefly, tissue was homogenized using a Teflon and glass homogenizer, with 20 volumes of Tris buffer (Tris 50 raM, pH 7.7, containing 100 mM NaCi and 1 mM CaCI,) and centrifuged at 48,000 x g for 15 min at 0°C. The pellet (membrane fraction) was re-suspended in a Polytron at 50% pulse setting for 12 min. The binding assay was carried out for 150 rain in a final volume of ! ml of Tris buffer containing 25/~1 of the membrane fraction (corresponding to 100-125/zg of protein) in the presence of phorbol-12,13-dibutyrate [20-'~H(N)] lAmersham, 18.4 Ci/mmol). Binding was terminated by rapid filtration through Whatman GF/C filters presoaked for 15 min in 0.5% polyethylcnimine. Filters were washed 3 times with 3 ml Tris 50 mM, pH 7.'/, and the radioactivity determined by liquid scintillation counting using Budget Solve (RPI), Specific binding was defined as the difference between total binding and that in presence of unlabelled 10 /zM PDBu dissolved in DMSO. Protein concentration was determined by the method of Lowry ct al. ,2. Coronal sections (20/zm) were taken in duplicates at 100 #m intervals throughout the septum on an AO cryostat and stained for Cresyl violet and acetylcholine,,,terase :4. A third series of sections through the septum (every 3(X) /~m) was processed for norepinephrine histofluorescence ~. All data were analyzed using the appropriate model analysis of variance (unweighted means ANOVA:SAS) followed by Duncan's multiple range test for post-hoe testing, When appropriate, Least Squares Means was employed for comparisons between groups. Six rats were excluded from the study because of improperly placed lesions or death. Data from the remaining 14 animals (CON: n = 5; MS/HSI: n = 5; MS/Gx: n = 4) were used for further analysis. Histological examination of the lesioned animals revealed
345 MS lesions that were typical of those produced in our laboratory 2. Thus, in the MS/HSI and MS/Gx groups there was total destruction of the medial septal region throughout the entire anterior-posterior extent. Destruction of the ventral limb of the diagonal band of Broca was also found in all lesioned animals, with variable destruction of the horizontal limb. In the CON group, limited gliosis was observed along the electrode track, but damage to the cholinergic neurons was absent. Peripheral NE fibers were observed on the cerebral arteries and choroid plexus in both the CON and MS/HSI groups, while these fibers were absent in the MS/Gx group. in the CON group, the binding affinity (K d) of [3HI PDBu was found to vary by hippocampal region (Table I). Thus, the Ko was significantly greater in the VH compared to DH [F(~.m= 21.70, P < 0.01)]. Medial septal lesions, regardless of the presence or absence of HSI, resulted in an increased K d in the DH compared to the DH of controls (P < 0.05; Duncan's). No differences were observed in the affinity of [3H]PDBu binding in the ventral hippocampus when the MS lesioned groups were compared to each other or to controls (Fig. IA). in controls, the number of phorbol ester binding sites (Bmax) in the DH and the VH were similar (Table !). In the dorsal hippocampus, Bin,~ was similar among all experimental groups, suggesting no effect of MS lesions or HS! on the number of binding sites. However, in the VH, B,,,,x was found to be significantly greater in the MS/Gx group when compared to both the MS/HSI and the CON groups, which did not differ from each other [F
