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.

Immunohistochemical investigation of gamma-aminobutyric acid ontogeny and transient expression in the central nervous system of Xenopus laevis tadpoles.

The ontogeny of the gamma-aminobutyric acid (GABA)-positive neurons in the brain of Xenopus laevis tadpoles was investigated by means of immunohistochemistry, using specific antibodies both against GABA and its biosynthetic enzyme, glutamate decarboxylase (GAD). The results obtained with the two antisera were comparable. The GABA system differentiates very early during development. At stages 35/36, numerous GABA-positive neurons were seen throughout the prosencephalon and formed two main bilateral clusters within the lateral walls of the forebrain that ran caudally toward the hindbrain. Other GABA-immunolabeled cell bodies, together with a conspicuous network of GABAergic fibers, were seen in the posterior hypothalamus. In the spinal cord, the lateral marginal zone was GABA-positive, as were Rohon-Beard neurons, interneurons, and Kolmer-Agdhur cells. A very rich GABA innervation was observed in the pars intermedia of the pituitary. At stage 50, plentiful immunopositive neurons and fibers were found in the telencephalic hemispheres, the diencephalon, and the mesencephalon (optic tectum and tegmentum). By stage 54, the number of GABA-immunoreactive neurons in the posterior hypothalamus had decreased, so that, at stage 58, there were very few GABA-labeled cell bodies in the dorsolateral walls of the infundibulum, despite a strong GABAergic innervation within the median eminence and the pars intermedia. From stage 58 to stage 66, the distribution pattern was very similar to that described in the adult X. laevis and in other amphibian species. These results point to transient GABA expression within the hypothalamus, possibly related to either 1) a naturally occurring cell death or 2) a phenotypic switch.

Distribution and ultrastructure of tachykinin-like immunoreactivity in the frog (Rana esculenta) spinal cord, notably, the dorsal horn.

Tachykinins are involved in pain transmission at the spinal level. In frog, at least four tachykinins [TK] have been isolated from the brain, but their organization in the dorsal horn of the spinal cord is still poorly known. We have reexamined TK distribution by immunocytochemistry using an antibody recognizing the sequence common to all tachykinins in the spinal cord and dorsal root ganglia of the green frog Rana esculenta. A dense tachykinin-like immunoreactivity (TK-LI) was observed in the dorsolateral fasciculus or Lissauer's tract running ventromedial to the entry of the dorsal root and in numerous small and medium-sized dorsal root ganglion cells showing a primary afferent origin for part of TK-LI of the dorsal horn. The observation of numerous cell bodies in the dorsal horn, in addition, suggested a local or propriospinal origin. One group of cells was localized at the entrance of the Lissauer's tract TK-LI fibers into the dorsal horn, and another group was localized in the upper dorsal horn, a region with a low density of TK-LI fibers. It was suggested that the latter group may correspond to neurokinin B. Electron microscopic examination of the Lissauer's tract showed numerous immunoreactive axons, some located at the center of glomerular-like arrangements, suggesting that the information brought by these fibers may be transmitted and most probably modulated before their entry in the dorsal horn. In conclusion, the functional organization of tachykinins in the frog spinal cord seems to be similar to that of mammals, albeit with a different morphological organization.

Organization of tyrosine-hydroxylase immunopositive neurons in the brain of the crested newt, Triturus cristatus carnifex.

The localization of neurons, fibers, and terminals containing tyrosine hydroxylase (TH)-like immunoreactivity was studied in the brain of the crested newt by using an antiserum to rat phaeochromocytoma tyrosine hydroxylase. Immunoreactive cells and fibers were found in the spinal cord, the medulla oblongata (lateral periventricular areas), and the acousticolateral area. In the tegmentum mesencephali, two bilateral clusters of labelled cells were localized in the ventrolateral periventricular gray extending toward the caudal hypothalamus. In the hypothalamic tuberal lobes, the TH-like reactive neurons, frequently of CSF-contacting type, lined the dorsal wall of the lateral infundibular recesses. A thick network of TH-like reactive nerve fibers and terminals was observed in the perivascular zone of the median eminence and in the adenohypophysial pars intermedia. A number of labelled cell bodies were also found in the dorsal thalamus (pars intercalaris diencephali), the paraventricular organ, and the ventral wall of the preoptic recess. In the telencephalon, immunoreactive innervation was identified in the striatum, together with immunopositive cell bodies in the olfactory bulbs. The pattern of organization of TH-immunoreactive systems in the newt showed, except for some peculiarities (e.g., the labelled cell bodies in dorsal thalamus), close similarities to the arrangement typical of mammals.

Immunolocalization of aromatic L-amino acid decarboxylase in goldfish (Carassius auratus) brain.

The distribution of monoamines (catecholamines and serotonin) in fishes has been previously studied by immunohistochemistry of both the monoamines themselves and their biosynthetic enzymes. But the distribution of neurons containing aromatic L-amino acid decarboxylase, an enzyme involved in the biosynthesis of both catecholamines and serotonin, has up to now not been investigated. In order to improve knowledge about the localization of aromatic L-amino acid decarboxylase, neurons containing this enzyme were mapped immunohistochemically in the goldfish brain. Furthermore, neurons bearing aromatic L-amino acid decarboxylase immunoreactivity have been compared with those containing tyrosine hydroxylase and serotonin immunoreactivities. Our results show that distribution of aromatic L-amino acid decarboxylase immunoreactivity generally coincides with that of tyrosine hydroxylase and serotonin. Nevertheless, the presence of nine D cell groups (containing aromatic L-amino acid decarboxylase but lacking both catecholamines and serotonin) and six groups of neurons which are aromatic L-amino acid decarboxylase-immunonegative but contain tyrosine hydroxylase, and might produce L-DOPA, have been revealed. The occurrence of both D cell groups and presumptive L-DOPA neurons in goldfish brain is discussed in relation to similar findings in fish and mammalian brain.

Pre- and postsynaptic localizations of the CB1 cannabinoid receptor in the dorsal horn of the rat spinal cord.

Several lines of evidence show that endogenous and exogenous cannabinoids modulate pain transmission at the spinal level through specific cannabinoid-1 (CB1) receptors. Since anatomical data concerning spinal CB1 receptors are rather contradictory, we studied the cellular and subcellular localizations of the CB1 receptors by immunocytochemistry. Results show a dual pre- and postsynaptic localization of CB1 receptors. Presynaptic receptors are evidenced by the labeling of (1) heterogeneous dorsal root ganglion neurons and (2) axons of Lissauer's tract. Postsynaptic receptors are shown by the labeling of numerous interneurons in the outer part of lamina II. Double immunolabelings show that lamina II outer CB1 neurons, probably islet cells, may also contain GABA or nitric oxide synthase. Numerous CB1-containing neurons in lamina X are also immunostained with anti-nitric oxide synthase (NOS) antibody. Under the electron microscope, CB1 immunoreactivity is exclusively localized postsynaptically in both somatic and dendritic compartments. The absence of labeling on primary afferent axon terminals is discussed and compared to the absence of labeling on terminals or vesicle-containing dendrites of islet cells, where a presynaptic localization was expected according to data of the literature.

Topography of cholinergic and substance P pathways in the habenulo-interpeduncular system of the rat. An immunocytochemical and microchemical approach.

The topography of cholinergic and substance P containing habenulo-interpeduncular projections has been studied in the rat. The research has been carried out by combining choline acetyltransferase and substance P immunohistochemistry to experimental lesions and biochemical assays in microdissected brain areas. In addition, computer-assisted image analysis has been performed in order to obtain quantification of immunohistochemical data. The results show that cholinergic and substance P containing neurons have a different localization in the medial habenula and project to essentially different areas of the interpeduncular nucleus. Cholinergic neurons are crowded in the ventral two-thirds of the medial habenula while substance P containing cells are exclusively localized in the dorsal part of the nucleus. In most parts of the interpeduncular nucleus, choline acetyltransferase and substance P containing fibres and terminals are similarly segregated and no overlapping is apparent except for the rostralmost and the caudalmost ends of the nucleus. Cholinergic activity is largely concentrated in the central core of the nucleus, while substance P is preferentially localized in the peripheral subnuclei of the interpeduncular nucleus. In addition, both substance P and choline acetyltransferase levels show peculiar regional variations along the rostrocaudal axis of the interpeduncular nucleus. The results of experimental lesions demonstrate that the substance P projection carried by each fasciculus retroflexus is prevailingly ipsilateral in the rostral part of the interpeduncular nucleus and becomes progressively bilateral as far as more caudal regions of the nucleus are reached. By contrast, the cholinergic projections carried by each fasciculus retroflexus intermingle more rapidly and only show a slight ipsilateral dominance in the interpeduncular nucleus. The results of the study are discussed with reference to previous anatomical and neurochemical data which, in several instances, had given rise to discrepant interpretations.

Involvement of retinohypothalamic input, suprachiasmatic nucleus, magnocellular nucleus and locus coeruleus in control of melanotrope cells of Xenopus laevis: a retrograde and anterograde tracing study.

The amphibian Xenopus laevis is able to adapt the colour of its skin to the light intensity of the background, by releasing alpha-melanophore-stimulating hormone from the pars intermedia of the hypophysis. In this control various inhibitory (dopamine, gamma-aminobutyric acid, neuropeptide Y, noradrenaline) and stimulatory (thyrotropin-releasing hormone and corticotropin-releasing hormone) neural factors are involved. Dopamine, gamma-aminobutyric acid and neuropeptide Y are present in suprachiasmatic neurons and co-exist in synaptic contacts on the melanotrope cells in the pars intermedia, whereas noradrenaline occurs in the locus coeruleus and noradrenaline-containing fibres innervate the pars intermedia. Thyrotropin-releasing hormone and corticotropin-releasing hormone occur in axon terminals in the pars nervosa. In the present study, the neuronal origins of these factors have been identified using axonal tract tracing. Application of the tracers 1,1'dioctadecyl-3,3,3',3' tetramethyl indocarbocyanine and horseradish peroxidase into the pars intermedia resulted in labelled neurons in two brain areas, which were immunocytochemically identified as the suprachiasmatic nucleus and the locus coeruleus, indicating that these areas are involved in neural inhibition of the melanotrope cells. Thyrotropin-releasing hormone and corticotropin-releasing hormone were demonstrated immunocytochemically in the magnocellular nucleus. This area appeared to be labelled upon tracer application into the pars nervosa. This finding is in line with the idea that corticotropin-releasing hormone and thyrotropin-releasing hormone stimulate melanotrope cell activity after diffusion from the neural lobe to the pars intermedia. After anterograde filling of the optic nerve with horseradish peroxidase, labelled axons were traced up to the suprachiasmatic area where they showed to be in contact with suprachiasmatic neurons. These neurons showed a positive reaction with anti-neuropeptide Y and the same held for staining with anti-tyrosine hydroxylase. It is suggested that a retino-suprachiasmatic pathway is involved in the control of the melanotrope cells during the process of background adaptation.

CB1 cannabinoid receptors in amphibian spinal cord: relationships with some nociception markers.

The role of cannabinoids in spinal analgesia has so far been investigated in mammals and the interactions between cannabinoid receptors and markers involved in nociception have been described in the rat spinal cord. An endocannabinoid system is well developed also in the amphibian brain. However, the anatomical substrates of pain modulation have been scarcely investigated in anamniotes, neither is there reference to such a role for cannabinoids in lower vertebrates. In the present paper we employed multiple cytochemical approaches to study the distribution of CB1 cannabinoid receptors and their morphofunctional relationships with some nociception markers (i.e. Substance P, nitric oxide synthase, GABA and mu opioid receptors) in the spinal cord of the anuran amphibian Xenopus laevis. We found a co-distribution of CB1 receptors with the aforementioned signaling molecules, as well as a more limited cellular co-localization, in the dorsal and central fields of the spinal cord. These regions correspond to the mammalian laminae I-IV and X, respectively, areas strongly involved in spinal analgesia. Comparison of these results with those previously obtained in the mammalian spinal cord, reveals a number of similarities between the two systems and suggests that cannabinoids might participate in the control of pain sensitivity also in the amphibian spinal cord.

CB1-cannabinoid and mu-opioid receptor co-localization on postsynaptic target in the rat dorsal horn.

Cannabinoids and opioids interact in the control of nociception at the spinal level. Likely, several mechanisms are involved, with one of them being co-localization of cannabinoid and opioid receptors. In order to validate this hypothesis, a double labeling study of CB1 cannabinoid receptors and mu-opioid receptors in the dorsal horn of the rat spinal cord was performed. A strong co-localization of CB1 and mu-opioid receptors was observed in lamina II interneurons at the ultrastructural level. The physiological consequences of the co-localization are discussed.

Confocal microscopy analysis of NPY and TH immunoreactivities in the hypothalamo-hypophysial system of the frog.

Co-expression of tyrosine hydroxylase (TH) and neuropeptide Y (NPY) in local circuits innervating the hypothalamo-pituitary complex of the green frog, Rana ridibunda, was investigated using simultaneous double immunohistochemical technique, aided by dual-channel confocal laser scanning microscopy. NPY and TH immunoreactivities were observed co-occurring within a discrete neuronal population located in the suprachiasmatic region. In other hypothalamic areas, NPY-immunoreactive (IR) perikarya were generally codistributed, but distinct from TH-IR cells. In the adenohypophysial pars intermedia, the overlap between the two markers was partial, demonstrating the existence of multiple neuronal sources for the inputs to the gland.

The neuroanatomic binding pattern of [125I]atrial natriuretic factor in the Japanese quail brain.

Application of quantitative autoradiography technique provided a discrete anatomical distribution pattern of the atrial natriuretic factor (ANF) in the Japanese quail, Coturnix coturnix japonica, brain. The highest binding levels of [125I]ANF were shown to occur in telencephalon areas, such as fasciculus diagonalis Brocae (232 fmol/mg protein), septum (194 fmol/mg protein) and olfactory bulb (153 fmol/mg protein), and in posterior sites, such as nucleus interpeduncularis (177 fmol/mg protein), while lower levels (> 51 < 87 fmol/mg protein) were found in the hypothalamic sites of the diencephalon. The similar ANF receptor density levels in some brain areas of the quail as well as both mammalian and non-mammalian species suggest that this peptide might be involved in osmoregulatory activities (at the brain level) and furthermore indicate a probable functional conservation of ANF in vertebrates.

Multiple sources of the pituitary pars intermedia innervation in amphibians: a DiI retrograde tract-tracing study.

Afferent projections to the pituitary pars intermedia were studied using the DiI tract-tracing technique in two amphibian species, the urodelan Triturus carnifex, and the anuran Rana esculenta. After DiI crystal application into the pituitary intermediate lobe, in both species cells were retrogradely labeled in the preoptic nucleus, in the supra- and retro-chiasmatic hypothalamus and in the brainstem (especially in the area indicated as locus coeruleus). The findings are discussed in relation to data on the neurochemical nature of the innervation of the pars intermedia in amphibians.

Aromatic amino acid decarboxylase (AADC) immunohistochemistry in vertebrate brainstem with an antiserum raised against AADC made in E. coli.

Aromatic L-amino acid decarboxylase (AADC) is involved in the biosynthesis of catecholamines and indolamines. AADC is present in the nervous system, in the chromaffin cells, and in non-neuronal tissues. We tested the capacity of a new polyclonal antibody, obtained by immunization of rabbits with a recombinant protein beta-galactosidase-AADC, to detect monoaminergic neurons in the brainstem as well as monoaminergic paraneurons in the adrenal medulla from goldfish, frog, skink, quail, and mouse. In the adrenal gland we found an immunoreactivity that was consistent with the distributions of the chromaffin cells previously reported. In the brainstem, groups of immunoreactive neurons and several labelled fibers were observed in the five species studied. The raphe region showed cell bodies and processes similar to those previously identified as monoaminergic by other authors. In addition, in medulla oblongata and isthmic tegmentum we found, in goldfish, skink, and quail, neuronal groups similar to mammalian D groups which contain AADC but are devoided of serotonin and catecholamines.

Gonadal-GABAergic interaction is an important factor involved in photoperiod-induced 2-[125I] iodomelatonin binding changes in the Japanese quail brain.

The type of mechanism(s) by which melatonin alone and/or through the intervention of other putative neurotransmitters is able to control circadian rhythms remains unresolved. Comparison of 2-[125I]iodomelatonin binding pattern in the brain of castrated and gonadally intact Japanese quail (Coturnix japonica), using quantitative receptor autoradiography, displayed that the combination of the intact gonadal condition and a long-day (16L:8D) photostimulatory schedule is responsible for major binding changes. In fact, high and low binding levels were obtained in the suprachiasmatic area and nucleus ectomamillaris (p < 0.01) and in the nucleus preopticus anterior and paleostriatum primitivum (p < 0.001), respectively. A gonadal modulatory role was not always evident in all brain areas as revealed by long-day photic cycles producing diminished (p < 0.01) binding levels in the anterior neostriatum and the nucleus rotundus of both castrated and gonadally intact animals, although elevated values were also found in the substantia grisea centralis (p < 0.05) of the same animals. Saturation binding studies revealed that gonadal and/or photic effects induce alterations in the number of binding sites, whereas the affinity constant varied only in some hypothalamic sites. Testing of GABAergic activity on 2-[125I] iodomelatonin binding levels showed that this inhibitory neurotransmitter was responsible for increasing low receptor values. Moreover, GABA-dependent influences were shown to be mediated via a GABAA receptor subtype since bicuculline (specific antagonist of this site) inhibited the elevated GABA-induced melatonin binding levels in the above brain sites of the gonadally intact quail exposed to both photoperiod cycles. Even in this case, melatonin binding changes were due to the variations in the number of binding sites. The apparent GABAergic-gonadal influence resulting in changes of the 2-[125I] iodomelatonin binding values, under the different photic conditions, provides evidences of other probable neural mechanisms that entrain circadian rhythmicity in neuroendocrine activities and in sociosexual behaviors.

Cytochemistry of neurotransmitters: from statical analysis to the understanding of dynamic processes.

In the past thirty years, cytochemical methods have allowed neuroscientists to identify and localize neuroactive molecules (neurotransmitters and neuropeptides), their receptors and their synthetic enzymes, and have advanced the understanding of many neuronal functions. Classic methods (histochemical and immunohistochemical techniques) have been used extensively to draw neurochemical maps in adult and developing nervous systems. As a consequence, many neuroactive molecules have become specific biochemical markers for neuronal systems. Double labelling techniques have greatly contributed to the discovery of the coexistence of two or more chemical compounds in the same cell. The in situ hybridization technique has recently become a productive addition to the tools available to the neuroscientist, especially when combined with immunocytochemistry to correlate mRNAs and protein expression. Even today, innovative roles for neurocytochemistry continue to be found. The newest approaches based on RT-PCR (reverse transcriptase-polymerase chain reaction) promise levels of sensitivity never reached before in in situ studies, and can provide simultaneous expression/functional data at the single-cell level.

Relationships between neuronal cell adhesion molecule and LHRH neurons in the urodele brain: a developmental immunohistochemical study.

Polysialic acid (PSA), a homopolymer attached to neural cell adhesion molecule (NCAM) is considered a major hallmark of vertebrate cell migration. We studied the distribution of PSA-NCAM by immunohistochemistry, during brain development, in two urodele amphibians, Pleurodeles waltl and the neotenic newt Ambystoma mexicanum. In both species a gradual increase of immunolabelling was observed throughout the brain from developmental stage 30 to stage 52. At the onset of metamorphosis, some differences became evident: in Pleurodeles immunostaining was gradually restricted to the olfactory system while in Ambystoma, PSA-NCAM maintained a more extended distribution (for example throughout the telencephalic walls) suggesting, for the brain of this latter species, a rather preserved neuronal plasticity. The aim of the present work was to correlate the above described PSA-NCAM-immunoreactivity (IR) with the distribution of luteinizing hormone-releasing hormone (LH-RH) containing neurons, which represent a well known example of neural elements migrating from the olfactory placode. LHRH-IR, undetectable till stage 30, was later found together with PSA-NCAM-IR in both the olfactory system and septo-hypothalamic areas. Such observations further support a role of PSA in providing a migration route toward the establishment of a part, at least, of the urodele LHRH system. The possible functional meaning of the LHRH-containing neurons localized between dorsal and ventral thalamus of Ambystoma, never reported before in this area, almost devoid of PSA-NCAM-IR, is discussed.

Brain natriuretic peptide (BNP)-like immunoreactivity in the central nervous system of the crested newt.

The distribution of brain natriuretic peptide (BNP)-like immunoreactivity (ir) was studied in the brain of a urodele amphibian, the crested newt Triturus carnifex Laur. BNP-like immunoreactive neurons were found mainly in the caudal hypothalamus (retro- and supra-chiasmatic areas) and in the preoptic area. A widespread innervation throughout the brainstem as far as the spinal cord was also observed. By double immunostaining (after section incubation with a-BNP and a-tyrosine hydroxylase-TH-antibodies), close topographical relationships between BNP-like and TH-like immunoreactive neurons within the hypothalamus were found.

Dihydronicotinamide adenine dinucleotide diaphorase in the central nervous system of the crested newt.

By using a histochemical procedure, the distribution of neurons containing dihydronicotinamide adenine dinucleotide diaphorase (NADPHd) was examined in the brain, retina and olfactory epithelium of the urodele amphibian Triturus carnifex. Positive nerve fibers and terminals were observed throughout the brain and cell bodies were seen within the telencephalon, optic tectum, brain stem, cerebellum and spinal cord. In the retina, NADPHd labeling was localized in the outer segment of photoreceptors and in some amacrine cells as well as in the outer and inner plexiform layers. In the olfactory epithelium, NADPHd labeling was found in the olfactory neurons. By comparing NADPHd distribution with nitric oxide synthase (NOS) immunoreactivity (using a polyclonal antiserum raised against mouse cerebellar NOS) it was found that NADPHd labeling and NOS immunoreaction patterns generally matched. The organization of NADPHd and NOS containing neurons in the central nervous system of the crested newt, which is simpler than in other vertebrates investigated, shows some peculiarities, such as the occurrence of NADPHD in the pinealocytes of the epiphysis, nucleus rubber of the brain stem and eminentia cerebellaris ventralis of the cerebellum.