Life 3cieacee Vol . 21, pp .37g-384, 1977 Printed in the U .S .A .
Pergamon Press
HOMOSEXUAL MOUNTING BEHAVIOR INDUCED IN MALE RATS AND RABBITS BY A TRYPTOPHAN-FREE DIET Walter Fretta, Giovanni Biggio and Gian Luigi Gesse Institute of Pharmacology, University of Cagliari, Italy . (Received in final form June 29, 1977)
Summary The ingestion of a diet containing all aminoacids but tryptophan causes a marked depletion of brain eerotonin, tryptophan and $-hydroxy-indoleacetic acid both in rats and rabbits, Thie effect persists for more than 24 hours, At the time of maximal eerotonin depletion, a marked increase in male to male mounting behavior both in rats and rabbits was observed . This response was prevented by the administration of $-hydroxytryptophan, the direct eerotonin precursor. This finding supports the hypothesis that brain eerotonin plays on inhibitory role in controlling male sexual behavior, Tryptophan-free diets may provide a non-pharmacological tool for studying the role of eerotonin in different types of behavior . A large number of studies in the last few years hâve reported that p-chlorophenylalanine (PCPA), a rather selective inhibitor of tryptophan hydroxylase, induces homosexual mounting behavior in male rate (under conditions in which this behavior does not normally occur and facilitates the copulatory behavior of male rats with females in estrus (for references see 1) . These results raised the crucial question of whether the aphrodisiac effect of PCPA is the consequence of the depletion of brain eerotonin ($-HT)~ since this fact would imply that this neurotransmitter plays an inhibitory role in controlling copulatory behavior in male rats, Recently, we have reported that the administration to rats, of an aminoacid mixture lacking tryptophan (Try) or a Tryfree diet results in a rapids selective depletion of brain $-HT and its main metabolite, $-hydroxyindoleacetic acid ($-HIAA) (2, 3) . The mechanism of this effect is the rapid removal of endogenoun Try from circulation, secondary to the increased incorporation of this aminoacid into proteins in liver and other tissues (see also the discussion), We considered that the Try-free diet , which effectively decrease brain $-HT synthesis, might be a useful mean to clarify whether the aphrodisiac effect of PCPA is causally related to the depletion of brain $-HT, 379
3B0
Tryptophaa-Free Diets/Homosexual Behavior
Vol . 21, No . 3, 1977
Materials and Methods Twenty male Sprague-DawTey rate weighing 300-3$0 g and 20 male New Zealand rabbits weighing 2,$ to 3,0 kg were used . They were housed one per cage in wire-bottom cages at room temperature of 2¢°C and reversed light-dark cycle (lights off 10 :00 to 22 :00), The animals had free access to water but they were trained to consume their normal daily food in a period of 3 hours : food was presented ad libitum at 11 :00 and r®moved at 14 :00 i,e, feeding occurred during the dark phase of the light-dark cycle s which is the time these rodents normally eat (4), The animals received a balanced diet (see below) for 2$ days, On the 26th day they were randomly divided into two equal groups of 10 animals each : one group ate the regular diet and the other a Try-free diet (see below) . Rate ate a mean of 10,2 g of regular diet and q,~ g of Try-free diet , Rabbits ate a mean of 210 grams of either diets, Six hours after food presentation s rats were put in groups of 4 (two control rats and 2 rate fed with the Try-free diet ) into observation cages and observed for one hour, Obviously the animals were properly marked to be easily recognized, After each behavioral test y the animals were reisolated and retested at 8 days interval, ßach rat was tested twice after the Try-free diet and twice after the normal diet, In the days between teats all animals received the balance diet, The same experimental design was used for rabbits, The behavioral observations were made under dim red lights in clear plastic cages with wire bottom, Observation cages for rata had 2$x$Ox2$ cm size s those for rabbits had 1$Oxl$Ox~O cm size, Behavior was recorded on a 12 channel event recorder, The same kind of food was used in the form of pellets both for rats and rabbits, The Try-free diet , had the following composition : grams per 100 g of food, dray weight : sucrose 34,0 ; starch 30 .$ ; zein 2,$ ; ammonium tartrate 2,2 ; gelatin 10,0 ; corn oil 10,0 ; glycine 3 .0 ; arginine 0,04 ; lysine 1,0$ ; histidine 0,22 ; methfonine 0,42 ; phenylalanine 0,¢$ ; leucine 0,19 ; isoleucine 0,40 ; threonine 0,32 ; valine 0,$6 ; salt mixture of Osborne and Msndel 4,0 ; choline chloride 0,1$ ; plus a complete vitamin integration per 100 g (A~ 2$0 Ui De 2$ Ui ßr 1$ mgi B1~ 0 .6 mg ; B2~ 1,3 mgi 86e O " 4 mg3 PABA~ $,$ 2,$ mg ; H~ 80 ug ; PP S mg ; B ~ ~ 1 ug ; K~ 100 ug ; inositol 100 mg), The balanced diet designed from values reported to maintain rate and rabbits adequately and to permit normal growth ($)~ had an identical composition except it contained 0,3 per cent of LTry in the place of an equivalent amount of starch, The effect of Try-free diet on brain $-HT metabolism was studied in a separate group of male animals kept under identical conditions as the animals used for the behavioral observations, Brain Try $-HT and $-HIAA were assayed as previously described (6),
3e1
Tryptophan-Free Diata/Ro77nsaxtsal Behavior
Vol . 21, No . 3, 1977
Results Biochemical study, As expected from previous results (3), the administration of the diet lacking Try caused a marked decrease in brain Try $-HT and $-HIAA both in rate and rabbits, These changes were maximal 6 hours after food presentation and the depletion persisted for more than 24 hours both in rate and in rabbits,
RAT
f00 a0 W w
so
C
40
u w 0
M
RA "" IT
700 a0
.
ao
i
0
. --~~
ao
s
a ~-^-"
FNT
D-0
/ N14A
o-~
ta NOYRi
40 b
ao
~ a~ ART " R
a FOOD
"
ta
i
a4
~R """ NTATION
Tltr
Effect of a Try-free diet on brain Try, $-HT and $-HIAA in the rata and rabbits, Animals were killed 3~ 6~ 12~ 24 hrs after food presentation . Each point +_ S,B, is the average of 30 rats and 4 rabbits . # P ~ 0,001 in respect to the animals that at same time ate the basal diet, On the other hand y the Try-free diet caused no significant changes in the level of brain catecholamines (results not shown) . Behavioral observations, As Table 1 shows, a large percentage (about ~0~) of rate fed with the Try-free diet displayed repeated episodes of mounting behavior similar to those observed with PCPA . On the contrary only 10~ of the rats fed with the balanced diet showed sexual excitation, Rat treated with the Try-free diet mounted indifferently rata fed with the same diet or control rate .
382
Tryptophan-Free Diets/Homo~exunl Behavior
Vol . 21, No . 3, 1977
Table 1 Effect of a Try-free diet on homosexual mounting behavior in male rats and rabbits Animals
Diet
Rats
Balanced
10
Try-free
~0
"
Try-free + $-HTP Rabbits "
~ of males mounting other males
0
Balanced
1$
Try-free
6$
Each value is the mean from 20 animals, To decide whether the sexual excitation was related to deficiency of $-HT P $-hydroxytryptophan ($-HTP) the direct precursor of $-HT P was injected (2$ mg/kg intraperitoneally) into 10 rats fed with the Try-free diet 30 min before the mating test, The administration of $-HTP increased $-HT content by about 1$0~ (results not shown) and suppressed the copulatory behavior in all the animals . Similar results were obtained in rabbits 6$ per cent the rabbits a fed with the Try-free diet displayed mounting behavior s while only 1$ per cent of the control animals showed such behavior, Conversely as previously reported (~) the administration of L-Try (1$0 mg~kg intraperitoneally) failed to inhibit the copulatory behaviour both in control animals and in animals fed with the Try-free diet , Rabbits fed with the Try-free diet mounted indifferently rabbits fed with either diets. Moreover some of the sexually excited rabbits when paired with a cat s repeatedly attempted to mount it, Discussion We have confirmed our previous finding that Try-free diets are a new tool for effectively depleting brain $-HT in the rat ; moreover that these diets are equally effective in rabbits, The mechanism by which these diets cause the fall in brain Try, with the consequent decrease in $-HT synthesis e is the rapid removal of endogenous Try from circulation by liver and other tissues to allow protein synthesis to proceed, This process is stimulated by the dietary amino acids as the principal mechanism for their disposal (8),
Vol . 21, No . 3, 1977
Tryptophan-Free Diets/FIomosexual Behavior
383
The data reported provide strong support to the hypothesis that brain $-HT plays an inhibitory role in controlling sexual behavior in rata and rabbits . In fact we obtained mounting behavior, similar to that stimulated by PCPA, with a non pharmacological means, acting through a completely different mechanism from that of PCPA, but having in common with this drug the ability of inhibiting $-HT synthesis and depleting brain 5-HT storeB . Moreover, the involvement of $-HT in the observed sexual stimulation is supported by the fact that the sexual response to the Try-free diet was suppressed by the injection of $-HTP, the direct precursor of 5-HT . Pharmacological manipulations which stimulate male to male mounting behavior usually stimulate also the complete pattern of copulatory behavior in rats, provided the experimental subjects are chosen among animals with low basal sexual activity (1) . It will be, therefore, important, both for a theoretical and practical point of view, to clarify whether Try-free diets influence the copulatory behavior of sexually sluggish male rats or rabbits with receptive females . In general Try-free diets might provide a new means for understanding the role of brain 5-HT in other physiological functions such as sleep, pain perception, some kind of aggressiveness, etc, References 1. 2. 3. 4. 5. 6. 7. 8,
G .L . Gessa and A . Tagliamonte, Life Sci ., ire, 425-436 (1974) . G,L . Gessa, G . Biggio, F . Fadda, G .U . Corsini and A. Tagliamonte, J . Neurochem,, 22, 869-870 (1974), G. Biggio, F . Fadda, P . Fanai, A . Tagliamonte and G .L . Gessa, Life Sci ., lie, 1321-1329 (1974) . A .N . Worden, W, Lane-Patter "The U .F,A .W, handbook on the care and management of laboratory animals" IV (Edited by Lippincott) London (1972), R, Jaquot and M. Vigneron "Le besoin azote" P 67-94 IT (Edited by M. Bon), Alimentation Equilibre Commentry, Paris (19$9) . A. Tagliamonte, G. Biggio, L . Vargiu and G,L . Gessa, Life Sci, 12, part II, 277-287 (1973) . G .L . Gessa and A . Tagliamonte "Sexual behavior" p, 120 (Edited by M, Sandler and G.L . Gessa) Raven Press N,Y. (1975), H,N. Munro "Mammalian Protein Metabolism" p . 326, 1V Academic Press N .Y, (1964) .
Pergamon Press
HOMOSEXUAL MOUNTING BEHAVIOR INDUCED IN MALE RATS AND RABBITS BY A TRYPTOPHAN-FREE DIET Walter Fretta, Giovanni Biggio and Gian Luigi Gesse Institute of Pharmacology, University of Cagliari, Italy . (Received in final form June 29, 1977)
Summary The ingestion of a diet containing all aminoacids but tryptophan causes a marked depletion of brain eerotonin, tryptophan and $-hydroxy-indoleacetic acid both in rats and rabbits, Thie effect persists for more than 24 hours, At the time of maximal eerotonin depletion, a marked increase in male to male mounting behavior both in rats and rabbits was observed . This response was prevented by the administration of $-hydroxytryptophan, the direct eerotonin precursor. This finding supports the hypothesis that brain eerotonin plays on inhibitory role in controlling male sexual behavior, Tryptophan-free diets may provide a non-pharmacological tool for studying the role of eerotonin in different types of behavior . A large number of studies in the last few years hâve reported that p-chlorophenylalanine (PCPA), a rather selective inhibitor of tryptophan hydroxylase, induces homosexual mounting behavior in male rate (under conditions in which this behavior does not normally occur and facilitates the copulatory behavior of male rats with females in estrus (for references see 1) . These results raised the crucial question of whether the aphrodisiac effect of PCPA is the consequence of the depletion of brain eerotonin ($-HT)~ since this fact would imply that this neurotransmitter plays an inhibitory role in controlling copulatory behavior in male rats, Recently, we have reported that the administration to rats, of an aminoacid mixture lacking tryptophan (Try) or a Tryfree diet results in a rapids selective depletion of brain $-HT and its main metabolite, $-hydroxyindoleacetic acid ($-HIAA) (2, 3) . The mechanism of this effect is the rapid removal of endogenoun Try from circulation, secondary to the increased incorporation of this aminoacid into proteins in liver and other tissues (see also the discussion), We considered that the Try-free diet , which effectively decrease brain $-HT synthesis, might be a useful mean to clarify whether the aphrodisiac effect of PCPA is causally related to the depletion of brain $-HT, 379
3B0
Tryptophaa-Free Diets/Homosexual Behavior
Vol . 21, No . 3, 1977
Materials and Methods Twenty male Sprague-DawTey rate weighing 300-3$0 g and 20 male New Zealand rabbits weighing 2,$ to 3,0 kg were used . They were housed one per cage in wire-bottom cages at room temperature of 2¢°C and reversed light-dark cycle (lights off 10 :00 to 22 :00), The animals had free access to water but they were trained to consume their normal daily food in a period of 3 hours : food was presented ad libitum at 11 :00 and r®moved at 14 :00 i,e, feeding occurred during the dark phase of the light-dark cycle s which is the time these rodents normally eat (4), The animals received a balanced diet (see below) for 2$ days, On the 26th day they were randomly divided into two equal groups of 10 animals each : one group ate the regular diet and the other a Try-free diet (see below) . Rate ate a mean of 10,2 g of regular diet and q,~ g of Try-free diet , Rabbits ate a mean of 210 grams of either diets, Six hours after food presentation s rats were put in groups of 4 (two control rats and 2 rate fed with the Try-free diet ) into observation cages and observed for one hour, Obviously the animals were properly marked to be easily recognized, After each behavioral test y the animals were reisolated and retested at 8 days interval, ßach rat was tested twice after the Try-free diet and twice after the normal diet, In the days between teats all animals received the balance diet, The same experimental design was used for rabbits, The behavioral observations were made under dim red lights in clear plastic cages with wire bottom, Observation cages for rata had 2$x$Ox2$ cm size s those for rabbits had 1$Oxl$Ox~O cm size, Behavior was recorded on a 12 channel event recorder, The same kind of food was used in the form of pellets both for rats and rabbits, The Try-free diet , had the following composition : grams per 100 g of food, dray weight : sucrose 34,0 ; starch 30 .$ ; zein 2,$ ; ammonium tartrate 2,2 ; gelatin 10,0 ; corn oil 10,0 ; glycine 3 .0 ; arginine 0,04 ; lysine 1,0$ ; histidine 0,22 ; methfonine 0,42 ; phenylalanine 0,¢$ ; leucine 0,19 ; isoleucine 0,40 ; threonine 0,32 ; valine 0,$6 ; salt mixture of Osborne and Msndel 4,0 ; choline chloride 0,1$ ; plus a complete vitamin integration per 100 g (A~ 2$0 Ui De 2$ Ui ßr 1$ mgi B1~ 0 .6 mg ; B2~ 1,3 mgi 86e O " 4 mg3 PABA~ $,$ 2,$ mg ; H~ 80 ug ; PP S mg ; B ~ ~ 1 ug ; K~ 100 ug ; inositol 100 mg), The balanced diet designed from values reported to maintain rate and rabbits adequately and to permit normal growth ($)~ had an identical composition except it contained 0,3 per cent of LTry in the place of an equivalent amount of starch, The effect of Try-free diet on brain $-HT metabolism was studied in a separate group of male animals kept under identical conditions as the animals used for the behavioral observations, Brain Try $-HT and $-HIAA were assayed as previously described (6),
3e1
Tryptophan-Free Diata/Ro77nsaxtsal Behavior
Vol . 21, No . 3, 1977
Results Biochemical study, As expected from previous results (3), the administration of the diet lacking Try caused a marked decrease in brain Try $-HT and $-HIAA both in rate and rabbits, These changes were maximal 6 hours after food presentation and the depletion persisted for more than 24 hours both in rate and in rabbits,
RAT
f00 a0 W w
so
C
40
u w 0
M
RA "" IT
700 a0
.
ao
i
0
. --~~
ao
s
a ~-^-"
FNT
D-0
/ N14A
o-~
ta NOYRi
40 b
ao
~ a~ ART " R
a FOOD
"
ta
i
a4
~R """ NTATION
Tltr
Effect of a Try-free diet on brain Try, $-HT and $-HIAA in the rata and rabbits, Animals were killed 3~ 6~ 12~ 24 hrs after food presentation . Each point +_ S,B, is the average of 30 rats and 4 rabbits . # P ~ 0,001 in respect to the animals that at same time ate the basal diet, On the other hand y the Try-free diet caused no significant changes in the level of brain catecholamines (results not shown) . Behavioral observations, As Table 1 shows, a large percentage (about ~0~) of rate fed with the Try-free diet displayed repeated episodes of mounting behavior similar to those observed with PCPA . On the contrary only 10~ of the rats fed with the balanced diet showed sexual excitation, Rat treated with the Try-free diet mounted indifferently rata fed with the same diet or control rate .
382
Tryptophan-Free Diets/Homo~exunl Behavior
Vol . 21, No . 3, 1977
Table 1 Effect of a Try-free diet on homosexual mounting behavior in male rats and rabbits Animals
Diet
Rats
Balanced
10
Try-free
~0
"
Try-free + $-HTP Rabbits "
~ of males mounting other males
0
Balanced
1$
Try-free
6$
Each value is the mean from 20 animals, To decide whether the sexual excitation was related to deficiency of $-HT P $-hydroxytryptophan ($-HTP) the direct precursor of $-HT P was injected (2$ mg/kg intraperitoneally) into 10 rats fed with the Try-free diet 30 min before the mating test, The administration of $-HTP increased $-HT content by about 1$0~ (results not shown) and suppressed the copulatory behavior in all the animals . Similar results were obtained in rabbits 6$ per cent the rabbits a fed with the Try-free diet displayed mounting behavior s while only 1$ per cent of the control animals showed such behavior, Conversely as previously reported (~) the administration of L-Try (1$0 mg~kg intraperitoneally) failed to inhibit the copulatory behaviour both in control animals and in animals fed with the Try-free diet , Rabbits fed with the Try-free diet mounted indifferently rabbits fed with either diets. Moreover some of the sexually excited rabbits when paired with a cat s repeatedly attempted to mount it, Discussion We have confirmed our previous finding that Try-free diets are a new tool for effectively depleting brain $-HT in the rat ; moreover that these diets are equally effective in rabbits, The mechanism by which these diets cause the fall in brain Try, with the consequent decrease in $-HT synthesis e is the rapid removal of endogenous Try from circulation by liver and other tissues to allow protein synthesis to proceed, This process is stimulated by the dietary amino acids as the principal mechanism for their disposal (8),
Vol . 21, No . 3, 1977
Tryptophan-Free Diets/FIomosexual Behavior
383
The data reported provide strong support to the hypothesis that brain $-HT plays an inhibitory role in controlling sexual behavior in rata and rabbits . In fact we obtained mounting behavior, similar to that stimulated by PCPA, with a non pharmacological means, acting through a completely different mechanism from that of PCPA, but having in common with this drug the ability of inhibiting $-HT synthesis and depleting brain 5-HT storeB . Moreover, the involvement of $-HT in the observed sexual stimulation is supported by the fact that the sexual response to the Try-free diet was suppressed by the injection of $-HTP, the direct precursor of 5-HT . Pharmacological manipulations which stimulate male to male mounting behavior usually stimulate also the complete pattern of copulatory behavior in rats, provided the experimental subjects are chosen among animals with low basal sexual activity (1) . It will be, therefore, important, both for a theoretical and practical point of view, to clarify whether Try-free diets influence the copulatory behavior of sexually sluggish male rats or rabbits with receptive females . In general Try-free diets might provide a new means for understanding the role of brain 5-HT in other physiological functions such as sleep, pain perception, some kind of aggressiveness, etc, References 1. 2. 3. 4. 5. 6. 7. 8,
G .L . Gessa and A . Tagliamonte, Life Sci ., ire, 425-436 (1974) . G,L . Gessa, G . Biggio, F . Fadda, G .U . Corsini and A. Tagliamonte, J . Neurochem,, 22, 869-870 (1974), G. Biggio, F . Fadda, P . Fanai, A . Tagliamonte and G .L . Gessa, Life Sci ., lie, 1321-1329 (1974) . A .N . Worden, W, Lane-Patter "The U .F,A .W, handbook on the care and management of laboratory animals" IV (Edited by Lippincott) London (1972), R, Jaquot and M. Vigneron "Le besoin azote" P 67-94 IT (Edited by M. Bon), Alimentation Equilibre Commentry, Paris (19$9) . A. Tagliamonte, G. Biggio, L . Vargiu and G,L . Gessa, Life Sci, 12, part II, 277-287 (1973) . G .L . Gessa and A . Tagliamonte "Sexual behavior" p, 120 (Edited by M, Sandler and G.L . Gessa) Raven Press N,Y. (1975), H,N. Munro "Mammalian Protein Metabolism" p . 326, 1V Academic Press N .Y, (1964) .
