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The issue of complexity is more and more present in numerous domains of biological research, including aging research. In the present paper, based on a selective review of literature, we propose both a conceptual and a methodological framework to address age-related changes in functional complexity of the neurobehavioral system, presumably resulting from modifications of the coupling between cognitive and sensorimotor processes. In particular, after reviewing pioneering and more recent studies on aging and complexity in the neuromusculoskeletal system, we explore the possibility that an age-induced increase in the coupling between cognitive and sensorimotor domains could be captured by a stronger covariation of high-order variables, common to both cognitive and sensorimotor functioning. Our main assumption is that these variables could behave as neurobehavioral markers of aging in the neuromusculoskeletal system. The present approach markedly differs from other traditional approaches, which focused on process-specific variable correlates of chronological age, domain-by-domain, and task-by-task. It provides a coherent conceptual framework, a terminology, and a method for studying age-related coupling of cognitive and sensorimotor processes with the use of complexity and nonlinear dynamical systems theories.

Anatomical and pharmacological evidence suggests a role for substance P (SP) in the control of vasopressin secretion, but the origins of SP-immunoreactive (IR) projections to the paraventricular (PVH) and supraoptic (SO) nuclei of the hypothalamus have not yet been identified. Combined axonal transport, immunohistochemical, and ablation approaches were used to characterize the organization of SP-IR projections to the PVH. The results may be summarized as follows: (1) SP-IR projections are broadly and prominently distributed throughout the SO and both the magnocellular and parvicellular divisions of the PVH. The distribution within the PVH is quite uniform. (2) Combined retrograde transport-immunohistochemical analyses identified multiple potential sources of SP-IR inputs to the PVH. These included a number of hypothalamic cell groups, the laterodorsal and peduculopontine tegmental nuclei, and the rostral and caudal aspects of the ventrolateral medulla. Portions of the tegmental and medullary SP-IR neurons that were retrogradely labelled following tracer deposits in the PVH also stained positively for choline acetyltransferase or tyrosine hydroxylase, respectively. (3) To evaluate the distribution and prominence of medullary SP-IR projections to the PVH and SO, staining for SP and catecholamine-synthesizing enzymes was carried out in animals that had previously received knife cuts at the level of the pontomedullary border. Pronounced, and roughly parallel decrements in staining for peptide and amines were seen in the magnocellular division of the PVH and in the SO; less marked reductions in SP-IR were evident in the parvicellular division of the PVH. Collectively, these results suggest that SP-IR varicosities are in a position to influence multiple visceral regulatory cell types in the PVH and SO. Inputs to the magnocellular neurosecretory system arise in large measure from medullary neurons in which SP coexists with catecholamines. SP-IR projections to the parvicellular division of the PVH appear to originate from a number of sources.

The effects of chronic central administration of corticotropin-releasing factor (CRF) on food intake, body weight, and hypothalamic-pituitary-adrenocortical hormones were investigated in rats. The infusion of ovine CRF at doses of 0.3 and 1.0 microgram/h continuously induced decrease in food intake and a suppression of body-weight gain for 7 days. The inhibition of body weight gain induced by CRF could not be accounted for solely by a decreased food intake since the suppression of body-weight gain in CRF-infused rats was significantly greater than that observed in rats which received the same amount of food as the CRF-infused rats. The content of proopiomelanocortin (POMC) -derived peptides in the anterior lobe of the pituitary as well as the plasma levels of ACTH and corticosterone (B) were significantly elevated in CRF-treated rats, and the CRF content in the hypothalamus was significantly decreased. These results suggest that chronic intracerebroventricular (icv) administration of CRF stimulates the synthesis and secretion of POMC-related peptides in the pituitary and suppresses food intake accompanied by inhibition of body weight gain. The results are similar to clinical and laboratory findings observed in patients with stress-induced anorexia.

Several peptides are generated from prosomatostatin (proSS) in addition to somatostatin-14 (SS-14) and somatostatin-28 (SS-28). These are SS-28(1–12), proSS(1–76) and, as shown more recently, proSS(1–63) and antrin. Important variations in the proportion of these molecular forms are seen among different tissues and among different species. Processing of the precursor in the human brain yields minimal quantities of SS-28(1–12) and high levels of proSS(1–76), namely in cortical areas and in the bed nucleus of the stria terminalis. This is in contrast with findings in rat brain, where SS-28(1–12) is a predominant molecular form. Antrin, which corresponds to proSS(1–10), reaches its highest concentration in the antral portion of the stomach (117 ± 13 pmol/g wet weight), where it is found in secretory granules of delta cells. We observed an inverse relationship between levels of antrin and proSS(1–63) after chromatography of various tissue extracts. This suggests a precursor-product relationship between these two peptides.

The biochemical characterization and anatomical distribution of somatostatin binding sites were examined in the brain of the frog Rana ridibunda, and the distribution of the receptors was compared with the location of somatostatin immunoreactive neurons. The pharmacological profile of somatostatin receptors was determined in the frog brain by means of an iodinated superagonist of somatostatin, [125I-Tyr0,DTrp8]S-14. Membrane-enriched preparations from frog brain homogenates were shown to contain high-affinity receptors (KD = 0.78 ± 0.34 nM; Bmax = 103 + 12.7 fmoles /mg protein) with pharmacological specificity for [DTrp] substituted S14 and S28 analogs. The distribution of somatostatin-binding sites was studied by autoradiography on coronal sections of frog brain. Various densities of somatostatin receptors were detected in discrete areas of the brain. The highest concentration of binding sites was observed in the olfactory bulb, in the pallium, and in the superficial tectum. Moderate binding was observed in the striatum, amygdaloid complex, preoptic area, and cerebellum. Immunocytochemical studies of the distribution of somatostatin-28 (S28) related peptides were also conducted in the frog brain. Two antisera that recognize distinct epitopes of the somatostatin molecule have been used for immunohistochemical mapping of the peptide. Antiserum SS9 recognizes both S28 and somatostatin-14 (S14) and allowed the labelling of perikarya. Antiserum S320 recognizes the N-terminal fragment (1–12) resulting from enzymatic cleavage of S28. This latter antiserum, which does not cross-react with S28, stained mainly neuronal processes. At the infundibular level, however, both antisera stained cell bodies and fibers. Immunoreactive somatostatin-related peptides were detected in many areas of the frog brain. In the diencephalon, a heavy accumulation of perikarya and fibers was seen in the preoptic nucleus, the dorsal and ventral infundibular nuclei, and the median eminence. Immunoreactive perikarya were also observed in the telencephalon, especially in the pallium and in thalamic nuclei. Immunostained processes were detected in many telencephalic areas and in the tectum. There was good correlation between the distribution of somatostatin-immunoreactive elements and the location of somatostatin-binding sites in several areas of the brain, in particular in the median pallium, the tectum, and the interpeduncular nucleus. In contrast, mismatching was observed in the olfactory bulb, lateral pallium, and the cerebellum (which contained moderate to high levels of binding sites but virtually no somatostatin-immunoreactive fibers) and the hypothalamic areas (preoptic area and infundibular nuclei), in which a low concentration of binding sites but a high density of immunoreactive perikarya and fibers were detected. The present data provide the first pharmacological characterization and anatomical distribution of somatostatin-binding sites in the brain of a non-mammalian vertebrate species. The present results suggest that many of the major features of somatostatinergic systems described in mammals (e.g., the wide distribution of somatostatinergic neurons and somatostatin-binding sites; pharmacological characteristics of somatostatin receptors) had already arisen in tetrapod ancestors. Such findings suggest that both neuroendocrine and extrahypothalamic somatostatinergic systems have important functions throughout the vertebrate phylum.

We have isolated from extracts of ovine hypothalami two molecules characterized as somatostatin-28 and somatostatin-4–28 (referred to as somatostatin-25). They were reproduced by solid phase synthesis. In equimolar ratio and depending upon the experimental conditions, synthetic somatostatin-28 and somatostatin-25 are 3–14 times more potent than somatostatin-14 to inhibit the basal in vitro secretion of growth hormone or as stimulated by prostaglandin (PGE 2 ). In early studies in vivo, somatostatin-28 and somatostatin-25 are also more potent than somatostatin-14 in inhibiting the secretion of growth hormone acutely stimulated in the rat by injection of morphine; somatostatin-28 is also longer-acting than somatostatin-14. These results suggest that somatostatin-14, as originally isolated, is a biologically active fragment of a larger molecule of greater specific activity; it should be considered as another form of somatostatin with high biological activity present in some tissues and likely secreted by the tissues along with somatostatin-14 and possibly other somatostatin-peptides of diverse sizes.

Cysteamine administration to rats results in a marked depletion of hypothalamic somatostatin-14 (SS14) and a decrease of the potassium- evoked in vitro release of SS14 without a significant change in the content or release of somatostatin-28(1–12)-like immunoreactivity (SS28(1–12)-L1). Furthermore, cysteamine enhances the spontaneous release and markedly potentiates the potassium-evoked release of SS14 in the in vitro slice preparation. However, in vitro-administered cysteamine does not alter the spontaneous or potassium-evoked release of SS28(1–12)-LI. Immunohistochemical visualization of hypothalamic neuronal cell bodies and fibers following cysteamine administration shows a disappearance of the SS14 immunoreactive fibers and cell bodies with no apparent change in the SS28(1–12) immunoreactive fibers and cell bodies. These data suggest that, in rat hypothalamus, selective release of SS14 and SS28(1–12) can occur. The results are discussed in relation to possible sites of storage and release of the somatostatin- related peptides from synaptic nerve terminals.

By using immune sera which recognize one or more of the 3 peptides, somatostatin-14 (SS14), somatostatin-28 (SS28) and somatostatin-28(1-12) (SS28(1-12)) we have characterized their immunohistochemical distribution in the hippocampal formation. There exist at least two independent neuronal systems containing pro-somatostatin-related peptides: an intrinsic system of cells in the polymorphic layers which branch locally, and a dense terminal field in the molecular layer of the dentate gyrus that may constitute a portion of the entorhinal-dentate projection. In addition, SS28 is the dominant form present in cell bodies, whereas SS28(1-12) is preferentially localized in neuronal processes and terminals. We could not detect SS14 immunohistochemically.

The primary structure of the NH2-terminally extended somatostatins isolated from ovine hypothalamic extracts, one containing 28 residues and the other 25, has been determined. The structure of somatostatin-28 is Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH; the shorter one, somatostatin-25, has the same sequence as somatostatin-28 except that the first three NH2-terminal residues are deleted. The two peptides as isolated were found to be oxidized at the methionine residue to the methionine sulfoxide. Their structures were established by subjecting the native peptides to direct sequence analysis in a Beckman 890C sequencer and identifying the released phenylthiohydantoin derivatives by high-performance liquid chromatography. Their structures were confirmed by trypsin digestion and isolation of all the tryptic peptides, followed by amino acid analysis of the tryptic fragments. Moreover, some of the tryptic peptides were matched with their respective synthetic replicates on high-performance liquid chromatography.

Cleavage of the peptide bonds of preprosomatostatin at basic residues near the carboxyl terminus yields somatostatin-14, somatostatin-28, and somatostatin-28 (1-12). However, little is known about the molecular forms derived from the amino terminal portion of the precursor, even though this part of the prohormone is highly conserved through evolution. By using an antibody against the amino terminus of prosomatostatin, a decapeptide with the structure Ala-Pro-Ser-Asp-Pro-Arg-Leu-Arg-Gln-Phe, corresponding to preprosomatostatin (25-34), was isolated from the endocrine portion of the rat stomach, the gastric antrum. The antral decapeptide may represent a bioactive product generated from prosomatostatin after a monobasic cleavage similar to that involved in the formation of somatostatin-28. In fact, a monobasic cleavage requires two basic residues and a domain containing nonpolar amino acids such as alanine or leucine, or both.

The possible role of medullary somatostatin as a neurotransmitter involved in blood pressure regulation was examined. Blood pressure and heart rate were measured in pentobarbital anesthetized rats while pmol quantities of somatostatin-28 (SS28), SS28-(15–28) (somatostatin-14), SS28-(1–12) or SS28-1(1–10) were injected into the caudal nucleus tractus solitarii (NTS). Injection of artificial cerebrospinal fluid (ACSF; 500 nl/10 s) alone was followed by a small (2 mmHg) statistically insignificant decrease in blood pressure. SS28, SS28-(15–28) or SS28-(1–12) induced an immediate and statistically significant decrease in blood pressure as compared to vehicle alone. SS28-(1–10) had no significant effect. SS28 was tested at three doses. The lowest dose of SS28 (2.5 pmol) induced an 8 mmHg drop in blood pressure, the medium dose (25 pmol), an 11 mmHg drop and the highest dose (250 pmol) a 16 mmHg drop. The other peptide fragments were tested only at the higher 250 pmol dose. SS28-(1–12) induced a significantly (P < 0.01) less drop in blood pressure (10 mmHg) when compared to either SS28 or SS28-(15–28) (18–19 mmHg). The hypotensive and bradycardiac effects of SS28 were prevented by pretreatment with methylscopolamine (5 mg/kg i.p.) 10 min prior to SS28. The cardiovascular effects of intramedullary injected SS28 were also blocked with an ipsilateral NTS injection of yohimbine or tolazoline 10 min prior to SS28. These data indicate that the hypotension and bradycardia following the intramedullary injection of SS28 is mediated through the parasympathetic nervous system and that the C-terminal portion of SS28 and SS28-(15–28) is important for potency, but not obligatory, for efficacy. In addition, because two different adrenergic blocking agents can prevent the cardiovascular effects of somatostatin, central adrenergic mechanisms may be involved.

The synthesis by solid-phase methodology of two glycosylated analogs of somatostatin [Glc-Asn 5 ]-SS and [NAcGlc-Asn 5 ]-SS is described. These two analogs have been biologically tested on the secretion of pituitary growth hormone, pancreatic glucagon and insulin. The results show that glycosylation of somatostatin on the Asn 5 residue decreases by a hundred fold the inhibition activity on GH release when tested in vitro . In vivo , since the activity is similar to somatostatin the carbohydrates are probably removed by some enzymatic reaction and thus liberate the full activity of somatostatin.

A 41 amino acid peptide with high intrinsic corticotropin-releasing activity was isolated from 1000 bovine hypothalami by means of immunoaffinity chromatography, gel filtration, and two steps of reverse phase HPLC. The primary structure of the amino terminal 39 amino acids was characterized by gas phase sequence analysis. The sequence of the amidated car☐yl terminal dipeptide was established by digestion of the intact natural product with Staphylococcus aureus V8 protease, dansylation of the digest and comparative reverse phase liquid chromatography studies with the synthetic dansylated dipeptides Ile-Ala-NH 2 , Ile-Ala-OH, Ala-Ile-NH 2 and Ala-Ile-OH. The complete structure of the bovine corticotropin-releasing factor was established as: Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu- Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Asn-Asn-Arg-Lys-Leu-Leu- Asp-Ile-Ala-NH 2 using approximately 650 pmol of material.

The action of cysteamine on anterior pituitary hormone secretion was studied in vivo using conscious, freely moving male rats and in vitro using anterior pituitary cells in monolayer culture. Administration of 500 μg cysteamine into the lateral cerebral ventricles of normal rats caused the complete inhibition of pulsatile GH secretion for a minimum of 6 h. This treatment also significantly decreased plasma concentrations of LH for at least 6 h in orchiectomized rats, TSH in short-term (0.5 month) thyroidectomized rats, and PRL in long-term (6 months) thyroidectomized rats. The in vivo stimulation of GH, LH, TSH and PRL with their respective releasing hormones 60 min after administration of cysteamine was not different from the response observed in rats pretreated with saline except for PRL where cysteamine pretreatment significantly inhibited the expected PRL increase. In vitro, 1 mM cysteamine decreased basal and TRH stimulated PRL release while not affecting basal or stimulated GH, LH, TSH and ACTH secretion. These data demonstrate the dramatic and wide-ranging effects of cysteamine on anterior pituitary hormone secretion. This action appears to be mediated through hypothalamic pathways for GH, LH and TSH and through a pituitary pathway for PRL.

A highly specific radioimmunoassay for one of the putative adrenomedullary [Met]enkephalin precursors, BAM-12P (Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-OH), has been developed. The BAM-12P antibodies recognize the COOH-terminal fragment of the peptide from Arg7 to Glu12 and do not crossreact with [Met5]- or [Leu5]enkephalin or any of their COOH-terminal lysine or arginine extended analogs. Specificity for the COOH-terminal Glu-OH is suggested by the 100% crossreactivity with BAM-12P5-12 and 0.3% crossreactivity with BAM-12P5-12 amide. Using these antibodies, we have measured three forms of BAM-12P-like immunoreactivity in extracts of bovine adrenal medulla, of which the major form (greater than 90%) corresponds to BAM-12P by molecular weight. Extracts of bovine adrenal cortex contain 1% the amount of a BAM-12P-like material (Mr approximately 1400; 20 ng per gland), possibly due to crosscontamination with adrenomedullary tissue. The major form of BAM-12P-like material in extracts of bovine neurointermediate pituitaries is of higher molecular weight than authentic BAM-12P (Mr approximately 4000); the remaining material (10%) corresponds to BAM-12P by molecular weight. There is no detectable BAM-12P-like immunoreactivity in crude or purified extracts of bovine anterior pituitaries. Extracts of bovine hypothalamic tissues contain small amounts of BAM-12P immunoreactivity (approximately 2 ng per fragment) which can be detected as one molecular form corresponding to a 1400-dalton molecule. The results indicate that the enkephalin precursor found in the adrenal medulla also may be present in the pituitary and hypothalamus. Furthermore, the processing of this molecule appears to be tissue-specific.

Following the discovery of the growth hormone release-inhibiting factor somatostatin from extracts of ovine hypothalamus1, an N-terminally extended somatostatin of 28 amino acids has been identified in mammalian tissue2–6. The original peptide, somatostatin-14 (SS14), corresponds to the C-terminus of somatostatin-28 (SS28). Both SS28 and SS14 have biological activity3,7–9, occur in several rat brain regions10–15, are present in cell bodies and nerve terminals11–14,16 and can be released in vitro upon depolarization in a calcium-dependent manner16–19. Further, high-affinity binding sites were described for SS14, which also bind SS28 (refs 20–23). Recently, a dodecapeptide which corresponds to the N-terminus of somatostatin-28, somatostatin-28(1–12), has been characterized in rat hypothalamus24. Radioimmunological25 and immunohistochemical13 studies have indicated the presence of SS28(1–12)-like immunoreactivity in several cortical and subcortical regions of the rat brain. This peptide was found to be unevenly distributed with the highest concentration in the hypothalamus, and preferentially localized to dendritic and axonal processes and terminals. These observations suggest that SS28(1–12) may be a neurotransmitter. In this study, we describe a calcium-dependent release of a SS28(1–12)-like peptide from hypothalamic slices in vitro. This finding supports a neurotransmitter function for this peptide.

A peptide representing the major form of somatostatin-like immunoreactivity was isolated from 600 rat pancreata by using anti-somatostatin affinity chromatography, gel permeation chromatography and reverse-phase high-performance liquid chromatography (HPLC). The isolated peptide elutes with the same retention time as synthetic somatostatin-14 in isocratic HPLC and its amino acid composition is in agreement with that of the tetradecapeptide. We propose that the structure of the major rat pancreatic somatostatin is identical to that of somatostatin-14 characterized in other species.

Intracerebroventricular (icv) injection of cysteamine to rats produced a marked depletion of somatostatin-14-like immunoreactivity (LI) in all rat brain regions examined. The somatostatin-28 (SS28)-LI and SS28(1-12)-LI were generally not altered by the cysteamine treatment. Following subcutaneous injection of the drug similar depletions of hypothalamic SS14-LI was observed with no change in SS28-LI nor SS28(1-12)-LI. In vitro cysteamine significantly increased the basal release of SS14-LI and markedly potentiated the evoked release of SS14-LI from hypothalamic slices. At 10 mM cysteamine, enhanced release of SS14-LI from hypothalamic slices was still observed despite a marked depletion of tissue content of SS14-LI.

Subcellular distribution and binding characteristics of the three endogenous peptides somatostatin-14 (SRIF-14), somatostatin-28 (SRIF-28) and somatostatin-28(1–12) (SRIF-28(1–12)) derived from preprosomatostatin were investigated in the rat brain cortex. The three peptides are predominantly recovered from a crude mitochondrial pellet (P2), containing the pinched off nerve endings. Specific high affinity binding sites for 125 I -N- Tyr-SRIF -14 and 125 I -N- Tyr-SRIF -28 are present on pituitary and brain membranes. Under the same conditions, 125 I -N- Tyr-SRIF -28(1–12) binding is undetectable. Moreover, SRIF-28(1–12) does not displace 125 I -N- Tyr-SRIF -14 or 125 I -N- Tyr-SRIF -28 binding. SRIF-28 is more potent than SRIF-14 to displace 125 I -N- Tyr-SRIF -28 binding to brain and pituitary membranes, while both peptides are equipotent to displace 125 I -N- Tyr-SRIF -14 binding. Finally, the regional distribution of 125 I -N- Tyr-SRIF -14 and 125 I -N- Tyr-SRIF -28 binding sites in the brain is identical. In conclusion, the present results are consistent with a neurotransmitter and neurohormonal role for SRIF-14 and SRIF-28. The function of SRIF-28(1–12) in brain remains to be elucidated. Additionally, a differential role for SRIF-14 and SRIF-28 both in adenohypophysis and brain cannot be ascertained at the present time.

Bovine anterior pituitary cells in culture respond to synthetic ovine corticotropin-releasing factor (CRF) with concomitant release of immunoreactive adrenocorticotropin, β-endorphin and γ-melanotropin. Gel permeation chromatography shows that the major molecular forms of these peptides released into the culture medium are ACTH1–39 β-lipotropin and two large forms of γ3-MSH (Mr >8.000). The effect of CRF is dose dependent and the maximal stimulation (5 × 10−9M CRF) gives increases in immunoreactive ACTH (5 fold), β-endorphin (2 fold) and γ3-MSH (6 fold) that are not significantly different from those obtained with 10 mM isobutyrylmethyl xanthine (IBMX). The ED50 for the ACTH, β-LPH and γ3-MSH dose-response curves to CRF are 3 × 10−10, 3 × 10−10 and 2 × 10−10 molar respectively. CRF has no such effect on bovine intermediate lobe cells. The results suggest that CRF acts to release the post-translational products of pro-opiomelanocortin processing in the anterior pituitary without discrimination or selectivity.

Somatostatin-28 1 12 concentrations were measured in Huntington's disease (HD) postmortem tissue using a specific radioimmunoassay. Concentrations of immunoreactive somatostatin-28 1 12 were significantly increased in the caudate and putamen but were unchanged in cortical areas A9 and A17. Since somatostatin-28 1 12 terminates with the amino acids Arg-Glu-OH, we examined whether this dodecapeptide compound might exert a neurotoxic effect. Injections of somatostatin-28 1 12 into rat striatum showed no evidence of histologic damage.

We have examined rat brains by immunohistochemistry with an antiserum raised against BAM22P, a synthetic fragment of the adrenomedullary precursor of Leu- and Met-enkephalin, in order to determine whether preproenkephalin derived peptides are detectable in the central nervous system. BAM22P antiserum stained fibers, nerve endings and cell bodies in many areas of the brain, especially the striatum, the septum and the hypothalamus. Comparative topographical studies showed that neurons recognized by anti-BAM22P overlapped structures found immunoreactive for enkephalins in the present study. In addition, BAM22P antiserum stained cell bodies in cerebral cortex and in caudate putamen. The presence of BAM22P immunoreactivity in presumptive enkephalin neurons suggests that some brain enkephalins arise from a precursor identical or closely related to the preproenkephalin A found in the bovine adrenal medulla. This does not preclude the possibility of enkephalins also arising from other precursors molecules.

Using a sensitive immunohistochemical procedure, we have undertaken a detailed morphological characterization of the vasoactive intestinal peptide-positive (VIP-positive) neuron in the cerebral cortex of the rat. VIP-positive neurons are present in all regions of cortex, and are usually strongly bipolar, possessing long, radially directed processes with very limited branching in the tangential plane. The most extensive dendritic branching occurs in layers I and deep IV-superficial V, and the density of axonal varicosities is highest in layers II-IV. In the visual cortex, approximately 50% of the labeled cell bodies are in layer II and III and 80% of the labeled cell bodies are contained in layers I-IV (superficial 600 microns of cortex). In order to determine the density and 3-dimensional distribution pattern of these cells, we prepared serial tangential sections through the rat visual cortex, mapped the distribution of all labeled cells in each section on transparent acetate sheets, and compressed these superimposed maps. This analysis demonstrated that: (1) approximately 1% of the cortical neurons are VIP-positive, (2) their distribution is fairly uniform and statistically random, (3) there are no large areas (i.e. with a diameter greater than 100 microns) that lack a VIP-positive cell, (4) on the average, there is one VIP-positive cell per column of 30 microns diameter, and (5) the average nearest neighbor distance on the compressed display is 15 microns. Given the morphological characteristics of VIP-positive cells, these data indicate that each VIP-containing cell is identified with a unique radial volume, which is generally between 15 and 60 microns in diameter, and overlaps with the contiguous domains of neighboring VIP-positive cells. These morphological data support the notion that VIP-containing neurons play an important functional role within radially oriented columns of cerebral cortex.

Acid extracts from rat pancreas and hypothalamus were analyzed for the presence of the antigenic determinant corresponding to the NH2 terminus of somatostatin-28 (SS28), using an antiserum directed against amino acids 1 to less than or equal to 11 of the SS28 molecule. On gel permeation chromatography the majority of the immunoreactive material from each tissue extract eluted in one zone compatible with a peptide of 1250 daltons. Purification of this immunoreactive material by reverse-phase HPLC and cation-exchange chromatography yielded two immunoreactive peptides from each tissue extract. The amino acid compositions of both peptides in pancreas and hypothalamus correspond to the fragment 1-12 of SS28. The more hydrophobic peptide from each tissue coeluted with synthetic SS28-(1-12) on HPLC, while the other one coeluted with synthetic SS28-(1-12)-amide. We conclude that the prosomatostatin fragment Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-OH is present in both rat hypothalamus and rat pancreas.

γ-Melanocyte stimulating hormone (γMSH) is a possibly biologically active material discovered in the cryptic N-terminus of the pro-opiocortin precursor by recombinant DNA analysis of bovine pituitary mRNA. Well-characterized antisera to synthetic bovine γ-3MSH ( γ 3 MSH) were used to localize immunoreactive sites in sections of formaldehyde-fixed rat brain and pituitary by the indirect immunoperoxidase technique. Specificity for staining was established by absorption with the synthetic antigen peptides or their fragments; staining was not blocked by absorption with synthetic replicates of other natural peptides that contain redundant amino acid sequences, with those of γMSH such as corticotropin or β-MSH. The general patterns of staining within adenohypophysis, intermediate lobe, and central nervous system closely followed the previously described patterns of β-endorphin immunoreactivity. Corticotrophs, all intermediate lobe cells and neuronal perikarya in the ventro-basal hypothalamus exhibited immunoreactivity for γ 3 MSH as they do for β-endorphin. Furthermore, the general distribution of immunoreactive nerve fibers and terminals within the diencephalon and pons was quite similar to endorphin immunoreactivity patterns as well. In series of alternating sections, prepared for either γ 3 MSH β-endorphin immunoreactivity, the same specific terminal fields were found to exhibit very similarly shaped varicose axons and probable terminal bouton configurations. However, the density of the innervation by fibers exhibiting immunoreactivity for the two peptides varied among the common target areas. Furthermore, the perikarya exhibiting γ 3 MSH immunoreactivity were fewer in number, smaller in size, and more medially clustered than those exhibiting immunoreactivity for β-endorphin. These results demonstrate that γ 3 MSH also occurs in rat brain and in pituitary cells which were already known to contain endorphin immunoreactivity. However, γ 3 MSH-immunoreactive neurones may not be coexistent with all endorphin-immunoreactive neurons, and these cells project with varying intensity to common target fields. Such observations are in agreement with the proposal of different processing of a common precursor in different cells.

Mammalian brain contains 3 peptides related to the pro-somatostatin molecule: somatostatin-14 (SS14), the form originally identified from hypothalamic extracts, somatostatin-28 (SS28) and somatostatin 28 (1-12) (SS28 (1-12)). By using antibodies which selectively recognize one or more of these 3 somatostatin-related peptides, we have characterized their immunohistochemical distribution in neocortex. These somatostatin-related peptides have a specific laminar distribution in cortex and are differentially distributed such that SS28 is largely restricted to cell bodies, whereas SS28 (1-12) is preferentially localized in neuronal processes and terminals in a density which far exceeds that revealed by SS-14 immunoreactivity. These data suggest that there may be an intraneuronal transformation from a SS28-like peptide to a SS28 (1-12)-like peptide. In addition, the enriched distribution of nerve fibers containing the antigenic determinant, SS28 (1-12), strongly implies that somatostatin-related peptides constitute a major neurotransmitter system in neocortex. The morphological characteristics of this system are homologous with long projection pathways such as the cortico-cortical specific intrinsic systems as well as projections.

Acid extracts of murine hypothalamic and extra-hypothalamic rat brains were analyzed by specific radioimmunoassays for the presence of somatostatin-14, somatostatin-28 and somatostatin-28(1–12)-like immunoreactivity. Seven molecular forms were observed after gel permeation chromatography. In addition to somatostatin-14, somatostatin-28 and somatostatin-28(1–12), there were two peptides of 4,400 and 7,500 mol. wt. which contained the somatostatin-28(1–12) sequence with an extension towards the NH2-terminus of pro-somatostatin. Moreover, two other peptides of 6,000 and 9,500 mol. wt. were detected containing the whole somatostatin-28 structure. These results imply that the processing of brain pro-somatostatin involves a minimum of four cleavage sites and yields at least seven peptides.

Two neuropeptides reacting with antibodies directed against the C-terminal region of somatostatin 281–12 were purified to homogeneity from rat brain extracts. Amino acid analysis revealed that the larger peptide (8 kdaltons) consisted of 76 amino acids. Microsequencing established its aminoterminal structure as: Ala-Pro-Ser-Asp-Pro-Arg-Leu-Arg-Gln-Phe-X-Gln-Lys. The 8 kdalton somatostatin 281–12-like peptide corresponds to the whole prosomatostatin molecule without Arg-Lys-somatostatin-14. The smaller peptide (5 kdaltons) consists of 44 amino acids and is generated after cleavage of a Leu-Leu bond at position 56–57 of pre-prosomatostatin. Both 8 kdalton and 5 kdalton somatostatin 281–12-like peptides contain somatostatin 281–12 at their C-termini. The 4 most abundant neuropeptides derived from pre-prosomatostatin (pre-proSS) and presently characterized are: somatostatin-14, somatostatin 281–12, somatostatin 28 and pre-proSS25–100.

A peptide with somatostatin-like immuno- and bioactivity has been isolated from 1165 rat hypothalami by using antisomatostatin affinity chromatography, gel filtration and reverse-phase high-performance liquid chromatography. The isolated peptide is indistinguishable from synthetic somatostatin-14 with respect to chromatographic properties and amino acid composition. We therefore propose that rat hypothalamic somatostatin-14 is identical in structure to somatostatin-14 found in other species.

The ontogeny of the pituitary's responsiveness to synthetic rat corticotropin-releasing hormone (CRH) in the late prenatal and early postnatal periods of rats was studied by a superfusion system using whole pituitaries. A significant increase of immunoreactive beta-endorphin (IR-beta-Ep) secretion in response to 10(-10) M CRH but not to 10(-11) M CRH was observed in pituitaries from the 15th day of gestation, the earliest day that we tested, whereas 10(-11) M CRH stimulated IR-beta-Ep release from the pituitaries of 17.5-day-old fetuses. Dose-related IR-beta-Ep secretions induced by 10(-12) M to 10(-10) M CRH were observed in pituitaries of 19.5- and 21.5-day-old fetuses, and 1-, 3- and 9-day-old newborn pups. CRH stimulated not only IR-beta-Ep and IR-adrenocorticotropic hormone (ACTH) but also IR-alpha-melanocyte-stimulating hormone (IR-alpha-MSH) secretions from fetal pituitaries. The content of IR-CRH in the hypothalamic extract from 15-day-old fetus was 6.6 +/- 3.6 pg/hypothalamus (mean +/- S.E.M.) and it gradually increased to reach 212.7 +/- 20.3 pg/hypothalamus on the 21.5th day of gestation. However, the content of IR-CRH in the hypothalamus dramatically decreased just after birth and then rapidly increased again from the 5th day after birth. These data indicate that the responsiveness of corticotrophs to CRH is already present on the 15th day of gestation, when the content of IR-CRH in the hypothalamus is extremely low and that the amount of hypothalamic IR-CRH dramatically dropped for several days just after birth in rats.

The concept of a hypothalamic neurohumoral control for anterior pituitary secretion postulates the existence of a growth hormone-releasing factor (GRF) of neuronal origin that stimulates the pituitary gland to release growth hormone (GH). Such a compound has not yet been isolated and characterized from the brain, although there is extensive physiological and biochemical evidence for its existence (reviewed in ref. 2). However, a 44-amino-acid amidated peptide having the physiological properties of GRF as well as chemical similarities was recently isolated from a human pancreatic tumour that had caused acromegaly. Two shorter biologically active fragments of 40 and 37 residues were also isolated. The synthetic replicates of these human pancreas GRF (hpGRF) peptides specifically stimulate GH release in vitro and in vivo. Assuming similarity or identity between the putative hypothalamic GRF and the tumour-derived hpGRF, we have used immunohistochemistry to search for hpGRF-like immunoreactivity in the brain. We report here that antisera against the hpGRF1-40 peptide specifically stain neuronal cell bodies in the arcuate nucleus of the primate hypothalamus, with fibres projecting to the median eminence and ending in contact with portal vessels. This topography is characteristic of a neuronal system elaborating a releasing factor. These results provide evidence that hypothalamic GRF is very similar, if not identical, to hpGRF.

A potent and specific growth hormone-releasing factor (GRF) was recently isolated and characterized from a human islet cell tumour of the pancreas that caused acromegaly1–3. Antibodies raised against the synthetic replicate of this peptide have allowed the immunohistochemical identification of GRF-producing neurones within the primate central nervous system4. Such neurones are found mainly in the arcuate nucleus4–6 in human and monkey hypothalamus, suggesting that this nucleus is a primary source of GRF. We have further investigated this hypothesis by studying the anatomical organization of GRF neurones in rat hypothalamus, using an antibody raised against the recently characterized rat hypothalamic GRF7 in normal animals and in animals neonatally treated with monosodium glutamate (MSG), a treatment which results in the selective destruction of arcuate nucleus neurones8–10. We present here the results which show that GRF-producing neurones are located mainly in the arcuate nucleus of rats. MSG treatment results in the complete loss of GRF- immunoreactive cell bodies within this nucleus and provokes a selective disappearance of GRF-immunoreactive fibres in the median eminence. These results show that the arcuate nucleus is the origin of the GRF-containing fibres that project to the median eminence and establish the MSG-treated rat as an in vivo model for studying growth hormone secretion in the absence of neurohumoral GRF.