Vasoactive Intestinal Polypeptide Neurons Research Articles

Innervation of VIP-immunoreactive neurons by the ventroposteromedial thalamic nucleus in the barrel cortex of the rat.

We investigated the synaptic terminals of fibers originating in the ventroposteromedial thalamic nucleus (VPM) and projecting to the main input layers (IV/III) of the rat posteromedial barrel subfield. It was our aim to determine whether or not the subpopulation of vasoactive intestinal polypeptide (VIP)-immunoreactive neurons in these layers are directly innervated by the sensory thalamus. Anterograde tracing with Phaseolus vulgaris leucoagglutinin (PHA-L) and immunohistochemistry for VIP were combined for correlated light and electron microscopic examination. Columns of cortical tissue were well defined by barrel-like patches of PHA-L-labeled fibers and boutons in layers IV and III. Within these columns VIP-immunoreactive perikarya were located mainly in supragranular layers. Marked perikarya were also seen in infragranular layers, but their immunoreactivity was often weaker. Granular layer IV, which is the main terminal field for thalamic fibers, contained fewer VIP neurons than supragranular layers. In the light microscope, however, PHA-L-labeled fibers appeared to contact the somata or proximal dendrites of 60-86% of the layer IV VIP neurons . By contrast, only 18-35% of the VIP neurons in the supragranular layers, which receive a moderately dense projection from the VPM, appeared to be contacted. PHA-L-labeled boutons were seen close to 13-25% of infragranular VIP-positive cells. Electron microscopy showed that thalamic fibers formed at most four asymmetric synapses on a single layer IV, VIP-positive neuron. Although the proportion of VIP-positive neurons with labeled synapses was lower in supragranular layers, most of them shared multiple asymmetric synapses with labeled thalamic fibers. Up to six labeled synapses were seen on individual VIP neurons in layer III. We conclude that subpopulations of VIP-immunoreactive neurons, located in layers IV, III, and II are directly innervated by the VPM. These neurons may be involved in the initial stages of cortical processing of sensory information from the large, mystacial vibrissae. Since VIP is known to be colocalized with the inhibitory transmitter GABA, it is likely that VIP neurons participate in the shaping of the receptive fields in the barrel cortex.

Suprachiasmatic nucleus of the human brain: an immunocytochemical and morphometric analysis.

The present paper describes the immunocytochemical and morphometric characteristics of two major cell groups of the suprachiasmatic nucleus (SCN) in the human hypothalamus: the vasopressin (VP) and vasoactive intestinal polypeptide (VIP) neuronal subdivisions. The dimensions (volume and length) and the number of neurons expressing each peptide in the two subdivisions were obtained, as well as the mean diameter of the cell nuclei. All morphometric parameters were studied in relation to sex and age. Brains of 42 human subjects (22 males and 20 females) ranging in age from 10 to 92 years were obtained at autopsy. The hypothalamic area containing the SCN was dissected from each brain, dehydrated, and embedded in paraffin. Serial sections of 6 microns were cut in a coronal plane and stained with thionin for general orientation. To determine the architectonic boundaries of the VP- and VIP-expressing cell populations every 25th section was immunocytochemically stained by means of antibodies against arginine VP or VIP using the peroxidase-antiperoxidase method. The VP- and VIP-expressing cell numbers in the SCN of each subject were estimated by unilaterally counting the number of nuclear profiles with the aid of a Zeiss microscope under x 500 magnification, using a deconvolution procedure and a correction for section thickness. The main portion of the VP positive neurons is located in the dorsomedial part of the SCN and is rostrocaudally longer in females than in males (1.76 +/- 0.12 mm and 1.40 +/- 0.10 mm, respectively). The volume of the VP subdivision is 0.244 +/- 0.017 mm3 and contains 6,890 +/- 520 VP-immunoreactive neurons, with a mean density of about 29,000 neurons/mm3. No significant sexual dimorphism or age-related alterations in the population of VP neurons is found. The VIP positive neurons are mainly located in the ventral and central part of the SCN and extend rostrocaudally in a similar way in females and males (1.07 +/- 0.08 mm and 1.02 +/- 0.11 mm, respectively). The volume of the VIP subdivision is 0.034 +/- 0.004 mm3 and contains 1,700 +/- 140 VIP-immunoreactive neurons, with a mean density of about 63,000 neurons/mm3. An age-dependent sexual dimorphism is observed in the number of VIP-expressing neurons in the SCN: young males have about twice as many VIP neurons as females of the same age, whereas in middle-aged subjects this sexual difference is reversed, and less robust, with females now having about 1.7 times as many VIP neurons as males. In old subjects the difference in VIP cell number between men and women disappears. The present study clearly shows that the population of VP neurons in the human SCN is considerably larger than the population of VIP neurons. Furthermore, the age-related sexual differences in the VIP cell number reinforces the idea that the SCN is not only involved in the timing of circadian rhythms but also in the temporal organization of reproductive functions.

Chapter 18 SCN output drives the autonomic nervous system: with special reference to the autonomic function related to the regulation of glucose metabolism

Publisher Summary This chapter describes the role of the suprachiasmatic nucleus (SCN) in the control of the autonomic nervous system with respect to the mechanism of glucose homeostasis. In the effort to elucidate the mechanism of circadian rhythms of food intake and energy metabolism using rats as experimental animals, the chapter finds evidence that the hypothalamic SCN has an important role in the regulation of glucose metabolism through the control of the autonomic nerves. When SCN vasoactive intestinal polypeptide (VIP) neurons receive signals from the circadian oscillator, they may transmit signals for a circadian rhythm to the autonomic functions that include mechanism to maintain glucose homeostasis. When these VIP neurons may receive signals of changes in outer and inner environments, they may produce signals for the alterations in the autonomic nerves and ensure survival, especially in the excitement of sympathetic nervous system related to the elevation of the blood glucose under stressful conditions.

Involvement of cerebral neurotensin in hyperglycemic response caused by 2-deoxy- d-glucose in rats

Previous studies have indicated that neurons containing a vasoactive intestinal polypeptide (VIP)-like immunoreactive substance (VIP neurons) in the suprachiasmatic nucleus (SCN) are involved in regulating glucose metabolism in rats. In this connection, it has been suggested that in rats, VIP neurons in the SCN have neurotensin (NT) receptors. To clarify the role of NT, we examined the effects of intracranial injection of NT and an NT-antagonist on the hyperglycemic response to intracranial injection of 2-deoxy- d-glucose (2DG) in rats. The hyperglycemic and hyperglucagonemic responses caused by intracerebroventricular injection of 2DG were significantly enhanced by intracerebroventricular co-injection of NT, but suppressed by co-injection of the NT-antagonist. Intraperitoneal injection of the NT-antagonist did not affect the hyperglycemic and hyperglucagonemic responses to 2DG. These results suggest that intracranial NT plays an endogenously enhancive role in the hyperglycemic and hyperglucagonemic responses caused by 2DG.

Expression of mRNA for the N-methyl- d-aspartate (NMDAR1) receptor and vasoactive intestinal polypeptide (VIP) co-exist in enteric neurons of the rat

Anatomical, physiological and pharmacological evidence suggests that vasoactive intestinal polypeptide neurons are important mediators of the descending inhibitory component of intestinal peristalsis. Pharmacological data also indicate that glutamate may modulate intestinal motility. Recently, we demonstrated that mRNA coding for the glutamate N-methyl- d-aspartate (NMDA) receptor is expressed by rat enteric neurons. In order to ascertain whether NMDA receptor message is expressed by vasoactive intestinal polypeptide (VIP) neurons, we employed a double in situ hybridization technique. We hybridized tissue sections from the stomach, ileum and descending colon of adult rats with an NMDAR1 oligoprobe and a VIP oligonucleotide. Enteric neurons expressing mRNA for both NMDA and VIP were found in the myenteric and submucosal ganglia at all of the sampling sites. These data suggest that the mechanism for glutamatergic excitation is present in VIP-containing enteric neurons.

A confocal approach to the morphofunctional characterization of the transient tyrosine hydroxylase system in the rat suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) of the neonatal rat is transiently innervated by tyrosine hydroxylase (TH) fibers of unknown origin and whose catecholaminergic nature is rather doubtful. In order to characterize this system morphofunctionally, immunocytochemical double labelling and confocal laser scanning microscopy analysis were employed on cryostat brain sections of 10-day-old rats. Simultaneous stainings for neuropeptide Y (NPY) and tyrosine hydroxylase (TH) immunoreactivity showed that they are not colocalized, neither in the SCN fibers nor in the intergeniculate leaflet (IGL) neurons, site of origin of the NPY projection to the SCN. Therefore, the possibility that SCN transient TH fiber system originates from the IGL could be excluded. Double labelling for TH and aromatic l-aminoacid decarboxylase (AADC) demonstrated that transient SCN TH immunoreactive (IR) fibers are AADC negative, thus supporting the hypothesis of their non-catecholaminergic nature. Moreover two new group of cells which are TH positive and AADC negative were found: one in the SCN and the other in the periventricular hypothalamic nucleus (PHN). The presence of somatostatin (SRIF) and TH in PHN neurons and SCN fibers suggested their possible colocalization, but double immunolabellings gave negative results. Simultaneous immunocytochemical staining for vasoactive intestinal polypeptide (VIP) and TH showed that TH fibers may interact with ventrolateral SCN VIP neurons. This result suggests a possible involvement of TH fibers in regulating VIP cells activity in the entrainment of circadian rhythms.

Dopaminergic defect of enteric nervous system in Parkinson's disease patients with chronic constipation

Summary Clinical studies suggest that gut disorders are common in Parkinson's disease, but the morphological basis is unknown. Depletion of dopamine-containing neurons in the central nervous system is a basic defect in Parkinson's disease. We compared colonic tissue from 11 patients with advanced Parkinson's disease, 17 with adenocarcinoma (normal tissue was studied), and five who underwent colectomy for severe constipation. Immunohistochemistry was used to stain myenteric and submucosal neurons for dopamine, tyrosine hydroxylase, and vasoactive intestinal polypeptide (VIP). Each class of neurons was quantified as a percentage of the total neuronal population stained for the marker protein gene product 9·5. Nine of the 11 Parkinson's disease patients had substantially fewer dopaminergic myenteric neurons than the other subjects (mean 0·4 [SE 0·2] vs 6·9 [2·3] in controls and 5·7 [2·0] in constipated subjects). There was very little difference between the groups in numbers of tyrosine-hydroxylase and VIP neurons. Two Parkinson's disease patients had similar distributions of all types of neurons, including dopaminergic myenteric neurons, to the controls. High-performance liquid chromatography showed lower levels of dopamine in the muscularis externa (but not mucosa) in four Parkinson's disease patients than in four controls (7·3 [5·1] vs 24·2 [4·6] nmol per g protein), but levels of dopamine metabolites were similar in the two groups. The identification of this defect of dopaminergic neurons in the enteric nervous system in Parkinson's disease may lead to better treatment of colorectal dysfunction in this disease.

Age related change in the vasoactive intestinal polypeptide-immunoreactive neurons in the cerebral cortex of aged rats

Recent studies have explored certain changes in neurons containing neuropeptides with aging. However, until now, the degree of vasoactive intestinal polypeptide (VIP)-containing neuronal cell loss in the aged CNS has not yet been established with certainty, and available data are often contradictory. Changes in the VIP-containing neurons with aging were demonstrated by immunocytochemistry. Major loss of VIP-immunoreactive neurons in the aged rat brain were observed in frontal cortex area 3, parietal cortex area 1, hindlimb area, temporal cortex area 1 and 2, monocular part of occipital cortex area 1, occipital cortex area 2, and retrosplenial cortex. Frontal cortex area 1 and 2, parietal cortex area 2, forelimb area, binocular part of the occipital cortex area 1, and the dentate gyrus were moderately decreased. The axis of the VIP neurons in the aged group showed an irregular orientation tendency, especially in layers II and III. These results indicate the involvement of a neuronal system containing VIP in the aging brain, and provide the first morphological evidence for the loss of VIP neurons in the cerebral cortex of the aged rat.

Immunohistochemical characterisation of sympathetic and parasympathetic pelvic neurons projecting to the distal colon in the male rat

The pelvic ganglia are mixed ganglia containing both sympathetic and parasympathetic neurons that receive spinal input via the hypogastric (lumbar cord) and pelvic nerves (sacral cord), respectively. A recent study has utilised immunohistochemistry against synaptophysin (a protein associated with small vesicles) to visualise the preganglionic terminals in these ganglia. By selectively cutting the hypogastric or pelvic nerves and allowing subsequent terminal degeneration, the populations of parasympathetic and sympathetic preganglionic terminals, respectively, can be visualised. The present study has used this method in conjunction with retrograde labelling of pelvic neurons from the distal colon and double label immunofluorescence against tyrosine hydroxylase and vasoactive intestinal polypeptide (VIP) to identify and characterise the sympathetic and parasympathetic neurons projecting to the distal colon from the major pelvic ganglia of the male rat. Approximately equal numbers of distal colonic-projecting pelvic neurons are sympathetic and parasympathetic. Almost all noradrenergic neurons are sympathetic. Of the VIP neurons that project to the distal colon approximately one third are sympathetic, one third parasympathetic and the remaining third are possibly innervated by both the lumbar and sacral cord. Extrapolation from our results also suggests that the majority of non-noradrenergic neuropeptide Y neurons (which are known to comprise the remainder of the neurons) are parasympathetic. These studies have demonstrated that the pelvic ganglia are a major source of sympathetic innervation to the distal bowel and have further shown that the distal colon is another target for the non-noradrenergic sympathetic neurons of the pelvic ganglia.

VIP neurons in the human SCN in relation to sex, age, and Alzheimer's disease

The brains of 46 control subjects and 21 Alzheimer's disease (AD) patients were studied to determine whether there are age-related or AD-related changes in the vasoactive intestinal polypeptide (VIP) neuron population of the human suprachiasmatic nucleus (SCN). The number of VIP expressing neurons in the SCN of females, ranging in age from 10–91 years, did not change during normal aging. In males, however, the number of VIP neurons in the SCN was highest in the young subjects (10–40 years of age), after which, a dramatic decrease occurred in middle-aged subjects. This resulted in an age-dependent sex difference in the VIP cell population of the SCN: young males had twice as many VIP expressing SCN neurons as young females, whereas in the middle-aged group, the females had twice as many VIP SCN neurons as the males. A significant decrease in the number of VIP expressing neurons in the SCN was found in female presenile AD patients, i.e., those younger than 65 years.

Vasoactive Intestinal Polypeptide‐containing Neurons and Processes in the Developing Hippocampus of Rats Prenatally Exposed to Methylazoxymethanol Acetate

AbstractDevelopmental alteration of the dendritic arborization and axonal boutons of vasoactive intestinal polypeptide (VIP) neurons in the CA1 hippocampus was immunohistochemically examined in both control and microcephalic rats at postnatal days 15 and 60. Microcephaly was induced by prenatal exposure to methylazoxymethanol acetate (MAM; 20 mg/kg) on day 15 of gestation (MAM rats). In the control at postnatal day 15, VIP‐immunoreactive axonal boutons were densely aggregated specifically in the stratum pyramidale. At postnatal day 60, these axonal boutons in the stratum pyramidale of the controls were somewhat less dense. Another aggregation of axonal boutons was also observed along the upper part of the stratum oriens near the alveus border (area A). In the MAM rats at postnatal day 15, on the other hand, VIP‐immunoreactive neurons in the CA1 hippocampus were decreased in number and the dendritic arborization of these neurons was very poor compared with the controls. The density of VIP‐immunoreactive axonal boutons was remarkably lower than the controls. In the MAM‐rats at postnatal day 60, density of the axonal boutons was almost similar with that of MAM‐rats at postnatal day 15 in the stratum pyramidale, whereas a drastic increase was observed in area A, which made the bouton aggregation more dense than in the controls at postnatal day 60. These observations suggest that there is an area‐dependent difference in the development of VIP neurons in the CA1 hippocampus which may be related to the area‐dependent heterogeneous vulnerability of VIP neurons to MAM exposure.

No changes in the number of vasoactive intestinal polypeptide (VIP)-expressing neurons in the suprachiasmatic nucleus of homosexual men; comparison with vasopressin-expressing neurons

In an earlier study we found more than twice as many vasopressin (AVP) neurons in the suprachiasmatic nucleus (SCN) of homosexual men as compared to heterosexual men. The present measurements in the same individuals showed that there is no difference in the number of vasoactive intestinal polypeptide (VIP)-expressing neurons in the SCN. The reduced nuclear diameter of both VIP and AVP neurons in the SCN as found in homosexual men points to metabolic alterations in the SCN in relation to sexual orientation.

Development of vasoactive intestinal polypeptide neurons in the human suprachiasmatic nucleus in relation to birth and sex

The development of vasoactive intestinal polypeptide (VIP) neurons was determined in the human suprachiasmatic nucleus (SCN) of 43 subjects ranging from 27 weeks of gestation to 30 years of age using immunocytochemistry and morphometry. VIP neurons were first observed at 31 weeks of gestation in the ventrolateral part of the SCN. From 3 months postnatally onwards, VIP positive neurons were observed in some subjects in the centromedial part of the SCN. The centromedial type of VIP staining became a constant finding only at 19 years of age, at term the SCN was still, very immature. Only in a few subjects some VIP neurons stained in the ventrolateral SCN and their number and nuclear diameter was small. Postnatally the number of VIP neurons increased gradually until around 3 years of age adult values were reached. After the age of 10 a clear sex difference in the number of VIP neurons was found: males having on average twice as many VIP neurons in the SCN as females. The adult VIP cell numbers in- the SCN amounted only 35% of those found earlier for vasopressin. The present data do not support a particular role for VIP neurons in those rhythms that are already present in early development, e.g., of the temperature rhythm in prematures of around 30 weeks gestational age. Our observations in this and earlier papers as well as animal studies do suggest though a possible role for VIP neurons in the SCN in sexual dimorphic functions such as reproduction and sexual behavior.

Distinct choline acetyltransferase (ChAT) and vasoactive intestinal polypeptide (VIP) bipolar neurons project to local blood vessels in the rat cerebral cortex

Innervation of rat intracortical cerebral blood vessels by acetylcholine (ACh) and vasoactive intestinal polypeptide (VIP) remains largely unexplored and it is known if the cells of origin are intra- or extracortical nor perivascular fibers colocalize ACh and VIP. Cortical cholinergic innervation arises primarily from the basal forebrain and to a small extent from intrinsic bipolar ACh neurons thought to be the sole source of cortical VIP. In order evaluate if intracortical perivascular ACh terminals could be distinguished from those of the basal forebrain by their colocalization with VIP, we performed a double immunofluorescence study and determined the percentage of colocalization of choline acetyltransferase (ChAT) and VIP in cortical neurons, as well as in terminal fields associated with intracortical blood vessels. From a total of 2103 cells examined in all cortical areas, VIP neurons accounted for the largest population (58.3%) followed by ChAT-positive cells (28.2%) with only 13.5% of cells being double-labelled for VIP and ChAT. Of the cortical ChAT-immunostained cells ( n = 878), 32.3% colocalized VIP whereas only 18.8% of VIP neurons ( n = 1509) also contained ChAT. In various cortical areas, ChAT cell bodies were seen to be contacted by VIP terminals which surrounded closely their cell soma and proximal dendrites. Perivascular fibers studied by double immunofluorescence and confocal microscopy were of three categories including cholinergic, VIPergic with a smaller population of fibers which costained for both ChAT and VIP. These results show hat cortical VIP neurons are much more numerous than those containing ChAT, and that a majority of VIP neurons do not colocalize with ChAT. This observation indicates that ACh and VIP are primarily located in distinct neuronal populations and that VIP cannot be used as a marker of intracortical ACh neurons and terminals. Our results further suggest that intracortical blood vessels are primarily under the influence of distinct ChAT and VIP perivascular fibers. Also, the presence of a subset of VIP and ChAT/VIP fibers in association with intracortical blood vessels strongly suggests a role for cortical bipolar neurons in local cerebrovascular regulation. The origin of the perivascular ChAT fibers which do not colocalize VIP, however, remains unknown.

Emergence of VIP rhythmicity following somatostatin depletion in the rat suprachiasmatic nucleus

Administration of a somatostatin (SS) depletor, cysteamine, markedly reduced SS levels in rat suprachiasmatic nucleus (SCN). At the same time, cysteamine administration induced a circadian rhythm of vasoactive intestinal polypeptide (VIP) content in the SCN, which otherwise remains constant under constant environmental conditions. These results suggest that the stable level of VIP in the SCN under constant conditions is not an intrinsic property of VIP neurons but a consequence of interactions with other components in the SCN.

NADPH diaphorase (nitric oxide synthase)-containing nerves in the enteropancreatic innervation: sources, co-stored neuropeptides, and pancreatic function.

Pancreatic ganglia are innervated by neurons in the gut and are formed by precursor cells that migrate into the pancreas from the bowel. The innervation of the pancreas, therefore, may be considered an extension of the enteric nervous system. NADPH-diaphorase is present in a subset of enteric neurons. We investigated the presence of NADPH-diaphorase in the enteropancreatic innervation, the contribution of extrinsic nerves to the NADPH-diaphorase-containing fibers of the gut and pancreas, and the coincident expression of NADPH-diaphorase NADPH-diaphorase in intrinsic neurons of these organs with neuropeptides. The possible role of nitric oxide in the neural regulation of pancreatic secretion was studied in isolated pancreatic lobules. Neuronal perikarya with NADPH-diaphorase activity were found in both Dogiel type I and type II neurons of the myenteric plexus of the stomach and duodenum. All galanin (GAL)-immunoreactive neurons and a small subset of vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-immunoreactive neurons contained NADPH-diaphorase activity. NADPH-diaphorase activity was also found in a subset of VIP and NPY-immunoreactive pancreatic neurons. Retrograde tracing with FluoroGold established that NADPH-diaphorase-containing neurons in the bowel project to the pancreas. NADPH-diaphorase-containing fibers were also found to be provided to both organs by neurons in dorsal root ganglia. Secretion of amylase was evoked by L-arginine. This effect was prevented by N(G)-nitro-L-arginine (L-NNA), which also inhibited VIP-stimulated secretion of amylase; however, L-NNA had no effect on amylase secretion stimulated by carbachol. These results provide support for the hypothesis that nitric oxide plays a role in the neural regulation of pancreatic secretion.

The diurnal rhythm in vasopressin V1a receptor expression in the suprachiasmatic nucleus is not dependent on vasopressin.

The mammalian suprachiasmatic nucleus is the site of the circadian rhythm generator. The degrees of expression there of several neuropeptides, including vasopressin and vasoactive intestinal polypeptide, follow a diurnal rhythm. The vasopressin V1a receptor, whose activation results in phosphoinositol hydrolysis and mobilization of intracellular calcium, is expressed in this nucleus. This study used double simultaneous hybridization histochemistry to show that V1a receptor transcripts are present in both vasopressin and vasoactive intestinal polypeptide neurons and that their levels follow a diurnal rhythm. Furthermore, the expression of the V1a receptor is 12 h out of phase from that of vasopressin. However, the receptor's diurnal rhythm is still maintained in the vasopressin-deficient Brattleboro rat, indicating that the V1a receptor's rhythm is independent of any vasopressin feedback.

Postnatal development of vasoactive intestinal polypeptide-containing neurons in the visual cortex of normal and dark-reared rats

The effects of dark rearing on the distribution and density of vasoactive intestinal polypeptide (VIP)-containing neurons in the visual cortical areas (17, 18 and 18a) of rats during postnatal development were examined immunohistochemically. Two groups of Wistar rats, one reared under normal lighting conditions and the other in complete darkness from birth, were used. VIP neurons showed a fairly similar distribution in the three visual areas, being predominantly present in layers II and III. Their pattern of development was found to be similar in the normal and dark-reared animals and was characterized by a marked increase from postnatal day (P) 7 to P21, followed by a gradual diminution to 24–31% of peak densities. Counts of labeled neurons at all ages examined showed that their density was similar in both groups at P7 and P14, but progressively greater in dark-reared animals from P21 and thereafter, so that they only fell to 38–43% of peak densities. Thus, by 60 days of age densities of VIP-labeled neurons in areas 17, 18 and 18a in dark-reared rats were 57%, 49% and 51% higher than in the corresponding areas of the age-matched control rats. These results indicate that the normal decline in the numbers of VIP neurons is not so marked under the conditions of dark rearing.

Immunohistochemical Examination of Developmental Brain Defects*

AbstractRecent advances in immunohistochemistry have revealed the precise distribution of various neurotransmitter systems, their projections and ultrastructure in the brain. Based on these data, the structure and function of specific neurons or a group of neurons in the brain can be understood in terms of the dynamics of the neurotransmitters they utilize. This strategy of neuroscience may be utilized to explore higher brain functions such as learning, memory, cognition and sleep. Prenatal exposure of pregnant rats to methylazoxymethanol acetate (MAM) on day 15 of gestation produces offspring with severe microcephaly (MAM‐rats). These MAM‐rats show various behavioral abnormalities in learning, memory and cognition as well as hyperactivity and reduction of paradoxical sleep. Developmental abnormalities of serotonin (5HT) fibers in the cerebral cortex and 5HT neurons in raphe nuclei are examined. The cell deficits of cortical target neurons and irregularly arranged cortical layers were observed. Developmental abnormalities of 5HT neurons are discussed in terms of target dependent secondary degeneration. Vasoactive intestinal polypeptide (VIP) has an important role in behavioral modification. VIP is suggested to have a functional correlation with 5HT in the brain. The reduction of VIP neurons and hypo‐aggregation of their axonal boutons were observed in the cerebral cortex and hippocampus of infant MAM‐rats. However, in adult MAM‐rats, VIP axonal boutons showed layer‐dependent hyper‐ or hypo‐aggregation compared with the age‐matched control. The difference in the number of VIP neurons became small between the controls and MAM‐rats. These immunohistochemical observations of brain defects have an important implication for understanding the abnormal behavior of MAM‐rats.

Neuronal associations in the rat suprachiasmatic nucleus demonstrated by immunoelectron microscopy.

The synaptic associations of neurons in the suprachiasmatic nucleus (SCN) of rats were examined by single immunolabeling for somatostatin (SRIH) and arginine vasopressin (AVP), and double immunolabeling for SRIH plus AVP and vasoactive intestinal polypeptide (VIP) plus AVP. Single immunolabeling showed that SRIH neurons, which displayed some somatic and dendritic spines, formed synaptic contacts with immunonegative and positive axon terminals. AVP neurons also formed synaptic contacts with both immunonegative and positive axon terminals. The immunonegative terminals contained small, spherical clear vesicles or flattened clear vesicles. A few immunopositive AVP fibers made synapses with immunonegative somatic or dendritic spines. Double immunolabeling showed synaptic associations between SRIH axons and AVP cell bodies or dendritic processes, and between AVP axons and the somata or dendrites of SRIH neurons. These findings suggest a reciprocal relation between the two types of neurons. Synaptic contacts between AVP neurons and VIP axon terminals were also demonstrated. Previously, we found synapses between SRIH axons and VIP neurons. Thus SRIH neurons appeared to regulate AVP and VIP neurons. On the basis of these findings, two possible oscillation systems of the SCN are proposed.