Endocannabinoids affect the reproductive functions in teleosts and amphibians.

Following the discovery in the brain of the bonyfish Fugu rubripes of two genes encoding for type 1 cannabinoid receptors (CB1A and CB1B), investigations on the phylogeny of these receptors have indicated that the cannabinergic system is highly conserved. Among the multiple functions modulated by cannabinoids/endocannabinoids through the CB1 receptors one of the more investigated is the mammalian reproduction. Therefore, since studies performed in animal models other than mammals might provide further insight into the biology of these signalling molecules, the major aim of the present paper was to review the comparative data pointing toward the endocannabinoid involvement in the reproductive control of non-mammalian vertebrates, namely bonyfish and amphibians. The expression and distribution of CB1 receptors were investigated in the CNS and gonads of two teleosts, Pelvicachromis pulcher and Carassius auratus as well as in the anuran amphibians Xenopus laevis and Rana esculenta. In general the large diffusion of neurons targeted by cannabinoids in both fish and amphibian forebrain indicate endocannabinoids as pivotal local messengers in several neural circuits involved in either sensory integrative activities, like the olfactory processes (in amphibians) and food response (in bonyfish), or neuroendocrine machinery (in both). By using immunohistochemistry for CB1 and GnRH-I, the codistribution of the two signalling molecules was found in the fish basal telencephalon and preoptic area, which are key centers for gonadotropic regulation in all vertebrates. A similar topographical codistribution was observed also in the septum of the telencephalon in Rana esculenta and Xenopus laevis. Interestingly, the double standard immunofluorescence on the same brain section, aided with a laser confocal microscope, showed that in anurans a subset of GnRH-I neurons exhibited also the CB1 immunostaining. The fact that CB1-LI-IR was found indeed in the FSH gonadotrophs of the Xenopus pituitary gland and CB1 receptors together with the fatty acid amide hydrolase, the degradative enzyme of the endocannabinoid anandamide, were demonstrated in both bonyfish and frog gonads, strongly suggests that endocannabinoids are involved in central and peripheral gonadotropic functions of teleosts and amphibians.

Immunocytochemical identification of growth hormone- and prolactin-producing cells in the hypophysis of the newt Triturus cristatus carnifex Laur.

Single indirect immunocytochemical methods (immunofluorescence, PAP, and ABC) and double sequential staining (ABC followed by immunofluorescence) were used to localize GH- and PRL-producing cells in the pituitary distal lobe from Triturus cristatus. The following antisera were employed: rabbit anti-ovine PRL, anti-Rana catesbeiana PRL, anti-ovine GH, anti-bovine GH, and monkey anti-rat GH. A cell population corresponding to type-2 acidophils localized in the dorsal and central region, under the intermediate lobe, immunoreacted with GH antisera. Both ovine and bullfrog PRL antisera labeled a large cell population, corresponding to type-1 acidophils, predominantly localized in the ventral anterior two-thirds of the gland. The pattern of localization shown by the two cell types, although consistent with the majority of data on adult amphibians, disproves the findings obtained on the same species by other authors.

Identification by immunofluorescence of ACTH-producing cells in the pituitary gland of the tree frog Hyla arborea.

The indirect immunofluorescence procedure was used to localize ACTH-producing cells in the pituitary distal lobe from Hyla arborea adult specimens; the antiserum employed was rabbit anti-synthetic ACTH (1-24) conjugated with BSA. Immunohistochemical staining was suppressed by solid-phase absorption of the anti-ACTH with the specific antigen. In the distal lobe one cell population is immunoreactive; these cells are predominantly localized in the anterior half, both ventrally and dorsally; they exclusively correspond to type-3 basophils. In the intermediate lobe the total cell population is immunoreactive. Parallel inspections carried out on the pituitary of Xenopus laevis specimens gave results fully consistent with those reported by other authors.

Identification by immunofluorescence of prolactin- and somatotrophin-producing cells in the pituitary gland of the tree frog Hyla arborea.

The indirect immunofluorescence procedure was used to localize prolactin (PRL)- and somatotrophin (STH)- producing cells in the pituitary distal lobe from Hyla arborea adult specimens. The following mammalian antisera were employed: rabbit anti-ovine PRL, antibovine PRL, anti-human PRL, anti-rat PRL, anti-ovine STH, anti-bovine STH, anti-human STH; monkey anti-rat STH. Immunocytochemical staining was suppressed by solid phase absorption of both anti-PRL and anti-STH with the specific antigen. Absorption of anti-PRL with STH and of anti-STH with PRL did not appreciably affect immunocytochemical staining. Treatment with the two antisera revealed two different reactive cell types, both acidophils. Using PRL antisera a strong fluorescence was found in the large acidophils located chiefly in the rostro-central and ventral areas of the distal lobe sagittal sections. A somewhat weaker fluorescence was observed using STH antisera in the fewer, small acidophils mostly concentrated in the dorso-caudal region and only sparsely scattered in the other areas of the pars distalis. Strikingly, the overall pattern of localization shown by the two cell types is similar to their already known distribution in the pituitary distal lobe of some other species of urodele and anuran amphibians.

Catecholaminergic drugs in chronic schizophrenia.

The effect of dopaminergic-related and stimulatory drugs have been studied in chronic hebephrenic schizophrenics untreated with neuroleptic drugs. 6 patients received therapy of 2 g L-dopa + 200 mg carbodopa per day orally for 30 days, then placebo for 30 days. Following that 3 of the same patients received therapy of 2 g L-dopa + 200 mg carbodopa + 300 mg imipramine orally for 30 days, then placebo for 30 days. Following that the same 3 patients received 1 mg apomorphine s.c. for 15 days, then placebo for 15 days, then 1 mg apomorphine s.c. + 2 g L-dopa + 200 mg carbodopa per os daily for 15 days, then placebo for 15 days. The patients were examined psychologically by the Wittenborn Rating Scale, the Weigl Object-Sorting Test, and tests for verbal learning and verbal association, before and after each therapeutic trial. Levels of FSH, LH, testosterone and GH were assayed radioimmunologically before, in the middle of and after each course of therapy. 2 patients showed improvement in the affective-behavioural symptomatology during therapy, while the other 4, who had a more severe degree of mental deterioration and destruction, were unchanged. FSH and LH levels, very low under basal conditions, did not change under therapy. Testosterone was very low before therapy and increased in only 1 subject. Normal basal GH levels increased during therapy in some of the patients, but not constantly. The results obtained are discussed in relation to the catecholamine hypotheses of schizophrenia.

Neuroendocrine effects of haloperidol therapy in chronic schizophrenia.

The neuroendocrine effects of haloperidol therapy have been examined in 62 male chronic schizophrenic patients, aged 16-62 years. The duration of the disease varied between 2 and 29 years. The patients were divided into 48 hebephrenics with onset of the disease at puberty, or immediately after puberty, and 14 paranoids with onset of the disease in adulthood. They received 6 mg i.m.p.d. of haloperidol, for 30 days, up to a total dose of 180 mg. The following hormonal variables were examined before therapy and at 10-20 and 30 days of treatment: total urinary gonadotropins, serum FSH and LH, GH response to insulin stimulation, ACTH reserve (Metyrapone test), total urinary 17-ketosteroids and 17-hydroxycorticoids before and after an ACTH stimulation test, serum testosterone, insulin response to glucose load, plasma thyroxine before and after a TSH stimulation test. The basic hormonal values revealed decreased secretion of total gonadotropins, FSH, LH, ACTH and testosterone, and increased insulin secretion. The haloperidol therapy seemed to stimulate the secretion of FSH, LH, total gonadotropins, ACTH and testosterone, up to normal or low-normal levels. No modifications were observed in the other hormonal variables. The significance of these results is discussed.

Immunocytochemical and ultrastructural study of Rana dalmatina PRL and GH pituitary cells during larval development.

Single and double simultaneous indirect immunogold methods and ultrathin sagittal sections of Rana dalmatina tadpole heads were used to follow cytodifferentiation of PRL and GH cells in the adenohypophysial primordium from developmental stage 26 to the end of metamorphosis. Rabbit anti-frog (Rana catesbeiana) PRL and rabbit anti-frog GH were employed in the single method, and rabbit anti-frog PRL and monkey anti-rat GH were used in the double simultaneous technique as primary antisera. At stage 27, a couple of cells with few small secretory granules immunoreactive with anti-frog GH are first identifiable. At stage 28, some cells with few membrane-bound secretory granules, 70-90 nm in diameter, and immunoreactive with anti-frog PRL and other cells with few secretory granules, 80-110 nm in diameter, and immunoreactive with anti-frog GH are present. As development progresses, differentiation in both cell types is shown by an increase in the cytoplasmic volume and in the number, size, and immunoreactivity of secretory granules. Neither comparison of adjacent ultrathin sections stained with the single labeling technique nor results of double simultaneous labeling showed the coexistence of PRL and GH within the same cell.

Prolactin secretion in chronic schizophrenia.

Basal prolactin secretion and its response to various stimuli have been studied in 20 chronic hebephrenic schizophrenics, 10 males and 10 females, aged 20-54 years. The duration of the disease varied between 4 and 30 years. Eight normal subjects from the hospital staff, four males and four females, matched for age, were used as controls. The patients had been off medication for 10 days in 17 cases, for 3 months in one case and for 1 year in two cases. The TRH stimulation test was done by giving 500 mug of TRH i.v., both to schizophrenics and controls. Schizophrenics and controls. Schizophrenics only were subjected to a 2-day therapy with chlorpromazine (4 mg/kg body weight per day orally), and therafter for 8 days to a combined therapy with chlorpromazine at the same dose plus 2-BRalpha-ergokryptine-mesilate (500 mg per day orally). Prolactin levels were assayed radioimmunologically in the basal condition, during the TRH stimulation test, after 2 days of chlorpromazine alone, and after 4 and 8 days of combined therapy with chlorpromazine plus 2-Br-alpha-ergokryptine-mesilate. The results obtained showed normal basal prolactin levels, significantly enhanced responses to TRH, normal increases after chlorpromazine alone, and substantial decreases after 2-Br-alpha-ergokryptine-mesilate. A possible relative catecholamine deficiency, related to the mental disease, is suggested to explain the results.

Glucose-insulin metabolism in herion addicts.

The insulinemic and glycemic response to a glucose load (100 g per os) was studied in 21 heroin addicts, 16 males and 5 females, age 16-28 years, history of addiction lasting from 6 months to 4 years with heroin alone (from 0.5 to 1.5 g/day i.v.). Nine normal sujects, from the hospital staff, 4 females and 5 males matched for age were use as control. The insulinemic and glycemic response to a glucose load was examined immediately after hospitalization, while the patients were still on heroin, 48 h later off drugs, and in 10 of the 21 cases 5 days later still off drugs. From the results obtained, it appears that in heroin addicts the glycemic response to the glucose load shows a delayed peak time. The insulin curves show increased insulin peaks, delayed peak time and prolonged hyperinsulinemia. The pathomechanism of heroin in inducing the above-metioned impairments is discussed, taking also in cosideration the possible influence of the drug on the neurotransmitter regulation of insulin.