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POU domain transcription factors are required for neuropeptide expression in selected subsets of hypothalamic neuroendocrine neurons. We now report that expression of the gonadotropin-releasing hormone (GnRH) gene, which controls sexual development, is regulated by the POU protein SCIP/Oct-6/Tst-1. Reverse transcriptase PCR cloning and RNase protection assays demonstrated the presence of SCIP/Oct-6/Tst-1 mRNA in the GnRH-producing neuronal cell line GT1-7. The physiological relevance of this regulatory activity was suggested by the detection of SCIP/Oct-6/Tst-1 mRNA in a subset of GnRH neurons in the hypothalamus of prepubertal female rats. Coexpression of SCIP/Oct-6/Tst-1 in neuronal cells inhibited rat GnRH (rGnRH) promoter activity via three regions of the proximal rGnRH promoter containing SCIP/Oct-6/Tst-1 binding sites. DNase I footprinting, gel shift assays, and DNA and protein mutagenesis studies indicated that both direct DNA binding and protein-protein interactions are required for SCIP/Oct-6/Tst-1 modulation of GnRH gene expression. Activation of SCIP/Oct-6/Tst-1 expression in terminally differentiated GnRH neurons may be a factor determining the ratio of phenotypically "inactive" versus "active" GnRH neurons during postnatal life.

The human cytomegalovirus (HCMV) major immediate-early promoter (MIEP) is one of the first promoters to activate upon infection. To examine HCMV MIEP tissue-specific expression, transgenic mice were established containing the lacZ gene regulated by the MIEP (nucleotides -670 to +54). In the transgenic mice, lacZ expression was demonstrated in 19 of 29 tissues tested by histochemical and immunochemical analyses. These tissues included brain, eye, spinal cord, esophagus, stomach, pancreas, kidney, bladder, testis, ovary, spleen, salivary gland, thymus, bone marrow, skin, cartilage, and cardiac, striated and smooth muscles. Although expression was observed in multiple organs, promoter activity was restricted to specific cell types. The cell types which demonstrated HCMV MIEP expression included retinal cells of the eye, ductile cells of the salivary gland, exocrine cells of the pancreas, mucosal cells of the stomach and intestine, neuronal cells of the brain, muscle fibers, thecal cells of the corpus luteum, and Leydig and sperm cells of the testis. These observations indicate that the HCMV MIEP is not a pan-specific promoter and that the majority of expressing tissues correlate with tissues naturally infected by the virus in the human host.

Purpose: To determine if tumor-specific monoclonal antibodies conjugated to superparamagnetic monocrystalline iron oxide nanoparticles can be used to yield specific diagnoses with the use of MR imaging., Methods: Monoclonal antibodies conjugated to monocrystalline iron oxide nanoparticles were given to nude rats with intracranial tumors either by intravenous injection, intraarterial injection with osmotic blood-brain barrier disruption, or direct intratumoral inoculation. Either L6, a tumor-specific antibody, or P-1.17, a control isotype-matched antibody, was used. Coronal T1-weighted, T2-weighted, and spoiled gradient-recalled acquisition in the steady state images were obtained before, 30 minutes after, 6 hours after, and 24 hours after injection., Results: Intravenous injection of greater than 2 mg of the tumor-specific antibody showed a specific pattern of enhancement of the tumors with the largest concentration of antibody in the area with the greatest density of tumor cells. The control antibody showed nonspecific changes. After intraarterial injection with barrier disruption to increase delivery globally or direct inoculation to increase delivery focally, no specific enhancement pattern was seen., Conclusion: Monoclonal antibodies conjugated with monocrystalline iron oxide particles may provide a method to obtain specific diagnoses with the use of MR imaging.

Neural transplantation of genetically modified cells has been successfully employed to reverse functional deficits in animal models of neurodegenerative disorders, including Parkinson's disease. While implanted PC12 cells secrete dopamine in vivo and can ameliorate dopamine deficiency in parkinsonian rat model systems, these cells either degenerate within 2-3 wk postimplantation (presumably due to the lack of neural trophic factor support at the site of implantation), or in some cases, form a tumor mass leading to the death of the host animal. To address these limitations, we have developed a genetically modified PC12 cell line that can synthesize nerve growth factor (NGF) under the control of a zinc-inducible metallothionein promoter. When implanted in the rat striatum and under in vivo zinc stimulation, these cells will neuro-differentiate, express tyrosine hydroxylase, and will undergo survival through potential autocrine trophic support. This regulatable cell line and general approach may provide additional insight on the potential utilization of cell transplants for treatment of Parkinson's disease and other neurodegenerative disorders.

Delivery of adenovirus, herpes simplex virus (HSV), and paramagnetic monocrystalline iron oxide nanoparticles (MION) to rat brain (n = 64) was assessed after intracerebral inoculation or osmotic disruption of the blood-brain barrier (BBB). After intracerebral inoculation, the area of distribution was 7.93 +/- 0.43 mm2 (n = 9) for MION and 9.17 +/- 1.27 mm2 (n = 9) for replication-defective adenovirus. The replication-compromised HSV RH105 spread to 14.00 +/- 0.87 mm2 (n = 8), but also had a large necrotic center (3.54 +/- 0.47 mm2). No infection was detected when virus was administered intra-arterially without hyperosmotic mannitol. After osmotic BBB disruption, delivery of the viruses and MIONs was detected throughout the disrupted cerebral cortex. Positive staining was found in 4 to 845 cells/100 microns thick coronal brain section (n = 7) after adenovirus administration, and in 13 to 197 cells/section (n = 8) after HSV administration. Cells of glial morphology were more frequently stained after administration of adenovirus, whereas neuronal cells were preferentially stained after delivery of both HSV vectors and MION. In a preliminary test of vector delivery in the feline, MION was detected throughout the white matter tracts after inoculation into normal cat brain. Thus MION may be a tool for use in vivo, to monitor the delivery of virus to the central nervous system. Additionally, BBB disruption may be an effective method to globally deliver recombinant viruses to the CNS.

The delivery of viral vectors to the brain for treatment of intracerebral tumors is most commonly accomplished by stereotaxic inoculation directly into the tumor. However, the small volume of distribution by inoculation may limit the efficacy of viral therapy of large or disseminated tumors. We have investigated mechanisms to increase vector delivery to intracerebral xenografts of human LX-1 small-cell lung carcinoma tumors in the nude rat. The distribution of Escherichia coli lacZ transgene expression from primary viral infection was assessed after delivery of recombinant virus by intratumor inoculation or intracarotid infusion with or without osmotic disruption of the blood-brain barrier (BBB). These studies used replication-compromised herpes simplex virus type 1 (HSV; vector RH105) and replication-defective adenovirus (AdRSVlacZ), which represent two of the most commonly proposed viral vectors for tumor therapy. Transvascular delivery of both viruses to intracerebral tumor was demonstrated when administered intraarterially (i.a.) after osmotic BBB disruption (n = 9 for adenovirus; n = 7 for HSV), while no virus infection was apparent after i.a. administration without BBB modification (n = 8 for adenovirus; n = 4 for HSV). The thymidine kinase-negative HSV vector infected clumps of tumor cells as a result of its ability to replicate selectively in dividing cells. Osmotic BBB disruption in combination with i.a. administration of viral vectors may offer a method of global delivery to treat disseminated brain tumors.

Phosphorylation of molecules involved in synaptic transmission by multifunctional protein kinases modulates both pre- and post-synaptic events in the central nervous system. The positioning of kinases near their substrates may be an important part of the regulatory mechanism. The A-kinase-anchoring proteins (AKAPs; ref. 3) are known to bind the regulatory subunit of cyclic AMP-dependent protein kinase A with nanomolar affinity. Here we show that anchoring of protein kinase A by AKAPs is required for the modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/kainate channels. Intracellular perfusion of cultured hippocampal neurons with peptides derived from the conserved kinase binding region of AKAPs prevented the protein kinase A-mediated regulation of AMPA/kainate currents as well as fast excitatory synaptic currents. This effect could be overcome by adding the purified catalytic subunit of protein kinase. A control peptide lacking kinase-binding activity had no effect. To our knowledge, these results provide the first evidence that anchoring of protein kinase A is crucial in the regulation of synaptic function.

Delivery of viral particles to the brain is limited by the volume of distribution that can be obtained. Additionally, there is currently no way to non-invasively monitor the distribution of virus following delivery to the central nervous system (CNS). To examine the delivery of virus-sized particles across the blood-brain barrier (BBB), dextran coated, superparamagnetic monocrystalline iron oxide particles, with a hydrodynamic diameter of 20 +/- 4 nm, were delivered to rat brain by direct intracerebral inoculation or by osmotic BBB disruption with hypertonic mannitol. Delivery of these particles was documented by magnetic resonance (MR) imaging and, unexpectedly, neuronal uptake was demonstrated by histochemical staining. Electron microscopy (EM) confirmed iron particle delivery across the capillary basement membrane and localization within CNS parenchymal cells following administration with BBB disruption. This is the first histologic and ultrastructural documentation of the delivery of particles the size of virions across the blood-brain barrier. Additionally, these dextran-coated, iron oxide particles may be useful, in and of themselves, as vectors for diagnostic and/or therapeutic interventions directed at the CNS.

The cAMP-dependent protein kinase (PKA) regulates a variety of diverse biochemical events through the phosphorylation of target proteins. Because PKA is a multifunctional enzyme with a broad substrate specificity, its compartmentalization may be a key regulatory event in controlling which particular target substrates are phosphorylated. In recent years it has been demonstrated that differential localization of the type II holoenzyme is directed through interaction of the regulatory subunit (RII) with a family of A-Kinase Anchoring Proteins (AKAPs). In this report, we review evidence for PKA compartmentalization and discuss the structural and functional properties of AKAPs.

The inner ear of the C3H/lpr autoimmune strain mouse was evaluated to identify potential mechanisms by which systemic autoimmune disease interferes with auditory function. The inner ears were immunohistochemically stained for IgG at ages before (2 months) and after (6-10 months) autoimmune disease onset and compared to age-matched nonautoimmune C3H/HeJ controls. Immunoreactivity for IgG was not seen in the 2 month C3H/lpr autoimmune mice or in either age group of the C3H/HeJ controls. On the other hand, all older C3H/lpr mice showed reaction product in the vessels of the cochlea, particularly the stria vascularis and bony capsule. Less frequent sites of staining were the geniculate ganglion, marrow cavities of the bony capsule, tensor tympani muscle, and on one occasion, a hair cell of the organ of Corti. These findings indicate that IgG is widespread within the cochlea and its vessels during systemic autoimmune disease and not directed against any specific sensorineural structure. This suggests a generalized or indirect mechanism whereby such systemic disease affects the inner ear.

Organotypic cerebellar cultures, some with incorporated portions of brainstem, were immunostained after 12-19 days in vitro with three different antibodies to tyrosine hydroxylase. Similar cultures were reacted with glyoxylic acid and examined for catecholamine histofluorescence. Locus coeruleus and other subcortical neurons were positive for tyrosine hydroxylase, as were Purkinje cells and outgrowth zone astrocytes. By contrast, only locus coeruleus neurons and their axons exhibited catecholamine histofluorescence after reaction with glyoxylic acid. These results confirm previously reported in vivo developmental studies indicating that tyrosine hydroxylase can be expressed in the absence of its normal biosynthetic products, and suggest that tyrosine hydroxylase cannot be considered to be a specific marker for catecholaminergic neurons in vitro, as well as during development in vivo.

Abstract An antiserum to neurohypophyseal prohormones was generated by immunization of rabbits with a synthetic peptide fragment bridging the prohormone cleavage site between the vasopressin (AVP) and human AVP-neurophysin sequences of pro-pressophysin. Polyclonal antibodies directed against this peptide cross-reacted with intact human pro-pressophysin (ED(50) of 260fmol), but not with either of the final products of enzymatic processing, AVP and human AVP-neurophysin. Gel electrophoresis and Western immunoblotting of pituitary or hypothalamic extracts from multiple species including mouse, cow and man identified a protein band of molecular weight consistent with intact pro-pressophysin; in hypothalamic extracts from normally-hydrated rats no protein bands were stained, but in extracts from Brattleboro rats a faint band in the area of pro-oxyphysin was identified. Immunohistochemical studies using the antiserum demonstrated the presence of only very small amounts of immunoreactive prohormone in a few widely scattered cells in the hypothalami of normally-hydrated rats. However, after 5 days of solute loading with 2% NaCl as drinking solution, staining for intact prohormone was prominent in the supraoptic and paraventricular nuclei of the hypothalamus. Combined immunoperoxidase-immunofluorescence labeling for prohormone and either AVP-neurophysin or oxytocin-neurophysin revealed prohormone staining in both types of magnocellular neurons in rat hypothalami. These studies suggest that during states of accelerated synthesis and secretion of neurohypophyseal hormones some accumulation of intact prohormone occurs in both AVP and oxytocin magnocellular neurons.

A monoclonal antibody (mAb L6) to a small-cell lung carcinoma surface antigen recognizes a common epitope of vasopressin-neurophysin and oxytocin-neurophysin in hypothalamic nuclei. We now report on the identification of a neurophysin-like precursor in human lung carcinoma (LX-1) cell membrane. mAb L6 immunoaffinity chromatography of solubilized membranes resulted in a single band of approximately 45 kDa. Western blot analysis demonstrated immunoreactivity of this band with mAb L6, anti-vasopressin, and an antibody to the vasopressin precursor, pro-pressophysin. N-terminal sequencing of this band demonstrated a 21-amino acid homology with the N terminus of human pro-pressophysin, and substitution of a Cys33 residue in the tumor antigen with Arg33. Absence of immunoreactivity with the antibodies described above in cytosolic extracts and culture medium suggests nonsecretion of processed or intact pro-pressophysin-like peptide. Northern analysis of LX-1 mRNA with a 30-mer to the C terminus of rat pro-pressophysin resulted in a band of approximately 1000 base pairs, 250 base pairs larger than hypothalamic message. In situ hybridization of LX-1 tumor-bearing nude rat brain with the same probe demonstrated specific hybridization in rat hypothalamus and xenografted tumor. These findings suggest expression of a pro-pressophysin-like protein in this tumor cell line that is preferentially targeted to the cell membrane.

A monoclonal antibody (mAb L6) to a carcinoma surface antigen has previously been shown to recognize neurophysins (NP), proteins associated with oxytocin and vasopressin. L6-reactivity in rat hypothalamus was confined to magnocellular neuronal systems. No staining was detected in parvicellular suprachiasmatic or paraventricular systems. mAb L6 immunoprecipitated vasopressin-neurophysin only under reducing conditions, and detected it in Western blots only after gel-renaturation and electroblotting in basic buffer. These findings suggest L6-reactivity to NP is conformation-sensitive, and imply NP expression in a unique configurational form in hypothalamic parvicellular systems.

Mouse monoclonal antibody (mAb) L6 identifies an antigen expressed on the cell surface of many different human carcinomas. While studying the binding activity of mAb L6 to intracerebral tumor xenografts of human lung carcinoma LX-1 cells in nude rats using immunohistological techniques, we observed that L6 can also bind to a cytoplasmic antigen expressed in the magnocellular component of the hypothalamo-neurohypophysial system. Double-labeling experiments with antisera to vasopressin and oxytocin confirmed the localization of L6 immunoreactivity within both peptide-containing cell groups. L6 immunoreactivity in Brattleboro rats (with genetic deletion in the vasopressin gene) was exclusively localized within oxytocin neurons. Oxytocin and vasopressin failed to block L6 staining which suggested that its target epitope resides within the neurophysin sequence, and this explanation was supported by the finding that adsorption of L6 with porcine neurophysin completely eliminated hypothalamic immunoreactivity. Western blot analysis of bovine neurophysin and human pituitary extracts identified L6-immunoreactive bands which corresponded to the position of neurophysin and pro-pressophysin, confirming that L6 immunoreactivity in hypothalamus is related to neurophysin. Thus, monoclonal antibody L6, which is highly reactive with a membrane antigen of human lung cancer cell line LX-1, recognizes a cytoplasmic epitope in hypothalamic neurons identified as neurophysin by immunohistochemistry and Western analysis.

The paraventricular nucleus of the rat hypothalamus has been shown to project to the medulla and spinal cord. The proportion of oxytocin-neurophysin (OTNP) axons to vasopressin-neurophysin (VPNP) axons in these structures is unknown. A major difficulty in resolving this problem in previous immunocytochemical studies was the lack of a specific antiserum to each rat neurophysin. In this study two approaches have been used: (1) comparison of immunostaining for neurophysin in normal versus homozygous Brattleboro rats with diabetes insipidus (HODI) which lack VPNP, and (2) application of an antiserum to both rat neurophysins absorbed with HODI rat hypothalamic-pituitary extracts which contain only OTNP. The latter would result in an antiserum specific for VPNP. Our results indicate that the axons which constitute the caudal projections from the paraventricular nucleus are predominately oxytocinergic, the vasopressinergic innervation being limited to the nucleus tractus solitarius, the dorsal motor nucleus of vagus, and the substantia gelatinosa. A similar number of reactive fibers were seen in the medulla and spinal cord of normal and HODI rats. No positive perikarya were observed caudal to the hypothalamus. Fibers in the medulla appeared to terminate in the nucleus of the solitary tract and in the dorsal motor nucleus of the vagus nerve. Positive fibers throughout the cord were present in the substantia gelatinosa and in the intermediolateral grey. The possible role(s) of these projections in integrating autonomic functions and afferent information with neuroendocrine regulation is discussed.

Neonatal administration of monosodium glutamate (MSG) produces necrosis of circumventricular structures, including perikarya in the medial-basal hypothalamus that contain beta-endorphin (BEND) and met-enkephalin. Since neonatal MSG treatment alters morphine analgesia, the present study examined neonatal MSG effects upon opioid analgesia observed following either BEND or d-ala d-leu enkephalin (DADL). Rats treated with either MSG or vehicle over the first ten post-natal days, were surgically prepared with a lateral ventricle cannula at 100 days of age. Respective groups received central injections of either BEND (0, 0.1, 0.5 or 1.0 microgram) or DADL (0, 4, 20 or 40 micrograms), and jump thresholds were assessed 15, 30, 45 and 60 min thereafter. Following testing, selected MSG-treated and control animals were prepared for BEND immunocytochemistry. While the magnitude, duration and sensitivity of BEND analgesia on the jump test failed to differ between groups, MSG-treated rats displayed a 10-fold leftward shift in sensitivity and a 200-300% increase in the magnitude of DADL analgesia. Immunocytochemical analysis indicated that MSG treatment depleted perikarya in the medial-basal hypothalamus, periventricular thalamic fibers and periaqueductal gray terminal fields that contained BEND. The differential effects of MSG treatment upon opiate and opioid analgesia are discussed in terms of possible alterations in opiate receptor subpopulations.

Immunohistochemical demonstration of glial fibrillary acidic protein (GFAP) was performed in human, sheep, rat and guinea pig pineal bodies to determine if there were species differences. Specialized "basket-like" arrangements of many GFAP-positive astrocytic processes were shown around sheep pinealocytes. Human pineals contained scattered astrocytic cell bodies and a moderate number of GFAP-positive astrocytic processes which, as in sheep, also surrounded pinealocytes, but without the dense basket-like arrangements. In both species GFAP-positive fibers were concentrated at the periphery of pseudolobules and around blood vessels. Rat and guinea pig pineals contained only rare astrocytic cell bodies and few GFAP-positive fibers throughout the glands, but had a concentration of parallel GFAP-positive fibers at the stalk. GFAP-positive fibers in human and sheep pineals may be derived from both intra- and extraglandular sites, whereas in rodents only rare processes appear to be derived from within the gland. Astrocytes may play a role in modulation of pineal indoleamines and norepinephrine, and the species differences observed suggest that this effect may be important in sheep and human pineals but not in rodents.

Both opioid peptides such as beta-endorphin and met-enkephalin and nonopioid peptides such as vasopressin and oxytocin increase pain thresholds in rodents. Antisera raised against each of these peptides have been developed for use in immunocytochemical and radioimmunoassay procedures. The present study assessed whether central administration of antisera raised against beta-endorphin (ABE), met-enkephalin (AME), arginine, vasopressin (AAVP) or oxytocin (AOT) altered tail-flick latencies elicited by three different levels of radiant heat, jump thresholds, core body temperatures and locomotor activity. ABE induced a transient hyperalgesia on the tail-flick test at thermal levels at which beta-endorphin administration would elicit an analgesic effect. While met-enkephalin increases tail-flick latencies elicited by high thermal levels, AME failed to alter latencies at this level, but rather induced a short-acting hyperalgesia at a low thermal level. While vasopressin increased tail-flick latencies at high thermal levels, AAVP produced reciprocal decreases. Yet AAVP inexplicably induced analgesic effects at moderate and low thermal levels. Finally, while oxytocin increased latencies at high thermal levels, AOT failed to alter latencies. Rather, it decreased latencies at the moderate thermal level and increased latencies at the low thermal level. Neither jump thresholds nor core body temperatures were affected by any antiserum pretreatment. While activity levels were unaffected by either ABE, AME or AAVP pretreatment, AOT decreased activity in a fashion complementry to oxytocin-induced hyperactivity and seizures. There data are discussed in terms of tonic versus phasic influences of these peptides upon pain perception.

Adrenocorticotropin and beta-lipotropin (beta-LPH) have been localized by immunoperoxidase methods in nerve cells and fibers of the hypothalamus and brain stem of the ewe. 6-mum sections were immunostained first for either ACTH or beta-LPH. The reaction products and the antibody complexes were then eluted completely from the tissue, and the same section was immunostained for the second peptide. Absorption of the primary antisera with a variety of peptide fragments of ACTH and beta-LPH demonstrated, immunocytochemically as well as by radioimmunoassay, that the ACTH and beta-LPH antisera were directed to the COOH- and NH(2)-termini of the peptides, respectively. Neither antiserum recognized any portion of the heterologous peptide. In the sequential staining procedure on the same tissue section, preincubation of the antisera with the homologous peptide abolished the staining, whereas preincubation with the heterologous peptide did not affect it, regardless of the order followed. Every nerve cell in the arcuate nucleus that contained ACTH also contained beta-LPH, but beta-LPH cells appeared, probably falsely, to be twice as numerous as ACTH cells. beta-LPH-positive fibers in and beyond the hypothalamus were also more numerous and stained more intensively than ACTH fibers. The salient exception was fibers in the infundibular zona externa, where the opposite was true. Our observations establish that ACTH and beta-LPH are contained in the same nerve cells They stongly favor biosynthesis in brain, probably from a common precursor molecule, as has been demonstrated in the pituitary gland. The complexity of the cytologic distribution pattern described suggests that the two peptides are not processed in the same manner by the nerve cell.

An immunoblotting method to detect low-molecular-weight peptides with monoclonal antibodies that normally fail to demonstrate immunoreactivity using conventional blotting techniques is described. Detection of neurophysin, insulin, calcitonin, vasopressin, and beta-endorphin electroblotted on nitrocellulose membranes was optimized after introducing four modifications into the conventional procedure. These include renaturing the gels after sodium dodecyl sulfate electrophoresis, electroblotting the renatured gels in basic transfer buffer, fixing and/or heating the blots, and using avidin/alkaline phosphatase conjugates for antigen/antibody detection. This technique likely enables the denatured peptides to regain their native conformation and, therefore, restores antigenicity and recognition by highly structural specific monoclonal antibodies. Although the most dramatic improvement with this technique is with monoclonal antibodies, a modest improvement in sensitivity can be obtained when immunoblots are probed with polyclonal antibodies. The high resolution of this system will be useful in probing blots of partial proteolytic digests of proteins with both monoclonal and polyclonal antibodies.

Purkinje neurons of the cerebellar cortex from a chemically and morphologically heterogeneous population containing some members that have gamma-aminobutyric acid (GABA), others that have immunoreactivity for motilin, and a small number that have both. The remaining 30-40% of all Purkinje cells have neither of these two neuroactive substances, leaving possibilities for other transmitter candidates. The evidence was compiled from double-staining immunocytochemical procedures performed on single sections of the cerebellum and brain stem in rat, mouse, and monkey. Two polyclonal antibodies were applied in succession, one directed against the midregion and COOH terminus of the 22-amino acid polypeptide motilin and the other against glutamic acid decarboxylase (glutamate decarboxylase; L-glutamate 1-carboxy-lyase, EC 4.1.1.15), the rate-limiting enzyme in the synthesis of the neurotransmitter GABA. The staining combinations employed the immunoperoxidase method, with different chromogens for distinguishing the motilin-like immunoreactivity from glutamic acid decarboxylase immunoreactivity by different colors, or the immunoperoxidase method for one antiserum and immunofluorescence for the other. The locations of both motilin and GABA cell types were mapped. The recognition of motilin in Purkinje cells calls for experimental definition of the role of this substance in the cerebellum and for reevaluation of the roles of Purkinje cells and of GABA in cerebellar function. The significant motilin representation in the flocculus, paraflocculus, and vermis suggests that it may be the Purkinje cell mediative chemical in the vestibular parts of the cerebellum. However, the presence of GABA as well in the same regions indicates that the chemical preference may be at least bimodal.

The distribution of adrenocorticotropin (ACTH) in monkey brain was examined by immunoperoxidase immunohistochemistry. An antiserum to ACTH that recognized the C-terminal portion of the molecule was used. Immunoreactive ACTH was visualized as an intraneuronal constituent with a widespread distribution throughout the brain. Reactive cell bodies were seen only in the region of the arcuate nucleus of the hypothalamus. Dense axonal networks were seen in the hypothalamus, mesencephalic gray, and in the region around the anterior commissure. No staining was seen in the cerebral cortex, cerebellum, hippocampus, or striatum. ACTH or fragments of ACTH may function as neurotransmitters or neuromodulators in primate brain.

Motilin-immunoreactivity was evaluated in rat brain using 15 different antisera and by combining gel filtration, high pressure gel filtration and reverse phase high pressure liquid chromatography with radioimmunoassay. Gel filtration chromatography demonstrated a high molecular weight and low molecular weight form of immunoreactive motilin. The high molecular weight form predominated in brain while the low molecular weight peptide was the predominant form of duodenum. The low molecular weight immunoreactive motilin was indistinguishable from synthetic porcine motilin by gel filtration and high molecular weight gel filtration. Low molecular weight rat motilin could, however, be distinguished from synthetic porcine motilin by high pressure liquid chromatography and certain antisera. Immunological results suggest that the slight structural difference may be in the N-terminal portion of the molecule. Immunoreactivity was measured in grossly and microdissected regions of the rat brain. The peptide had quite a unique distribution as highest concentrations are observed in the cerebellum. High concentrations were also observed in hypothalamic nuclei. Particularly high concentrations were noted in the organum vasculosum lamina terminalis. Lowest motilin concentrations in the rat brain were in the pons and the medulla. The distribution of motilin in rat brain suggests that it may have roles in regulating both neuroendocrine and neurological processes.