Diane T. Piekut

Adolescent development alters stressor-induced Fos immunoreactivity in rat brain

Adult-typical behavioural responses to environmental challenges as well as the stressor responsiveness of several neural systems emerge over adolescent development. The present study was undertaken to determine whether stressors might activate different neural populations in adult vs juvenile male rats. Fos-immunoreactivity was determined in various forebrain nuclei following 15 min or 2 h of restraint in 28- and 60-day-old male rats (representing late juvenile and young adult ages, respectively) and compared to non-restrained control animals at each age. Few Fos-positive cells were identified in unrestrained controls at either age. Restraint, however, induced the production of Fos in several areas. Fos immunoreactivity was marked in parvocellular regions of the paraventricular nucleus of the hypothalamus following both restraint periods and at both ages, an observation consistent with previous observations that restraint increases plasma corticosterone at both ages. And at both ages, Fos immunoreactivity was evident in magnocellular regions of the hypothalamus only following the longer restraint period. Fos immunoreactivity, however, clearly varied as a function of adolescent age in several regions. Moderate to intense Fos immunoreactivity was observed in adults in all divisions of the anterior olfactory nucleus, cortical and medial amygdaloid nuclei, pyriform cortex and tenia tecta. In contrast to the adult, only a few Fos positive cells were observed in any of these regions in juveniles. Exposure to the same stressor induced Fos in a broader spectrum of neurons in young adult than in juvenile male rats. The lack of Fos-positive cells in specific areas of juveniles may relate to maturation in specific amygdaloid nuclei, which project to many of the other regions that showed age-related differences in Fos production. The emergence over adolescence of Fos-positive cells in specific areas in response to stressors may underlie the emergence of adult-typical behavioural and neural stressor-responsiveness.

Distribution of Catecholamine-Synthesizing Enzymes in Goldfish Brains: Presumptive Dopamine and Norepinephrine Neuronal Organization

The organization of presumptive dopamine- and norepinephrine-synthesizing neurons in the brains of goldfish is described by using antibodies to tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) with avidin-biotin immunocytochemical techniques. In the hindbrain, TH-immunoreactive (IR) and DBH-IR cell bodies are located together in the following three regions: (1) dorsomedial medulla in the postobecular region, (2) medullary tegmentum from the level of the greatest expansion of the vagal lobes to the medullospinal transition, and (3) isthmal tegmentum dorsolateral to the medial longitudinal fasciculus. Elsewhere in the brain, TH-IR neurons were visualized in eight distinct forebrain neuronal groups; DBH-IR cell bodies were not observed. Fibers and terminals IR for TH and DBH were most dense in forebrain periventricular regions, i.e. adjacent to the third ventricle, and specifically around the lateral and preoptic recesses. In the telencephalon, a dense innervation of TH- and DBH-IR fibers was noted within the area dorsalis, pars lateralis and pars dorsalis. Within the area dorsalis, pars centralis TH-IR fibers were dense; DBH-IR fibers were not visualized in this region. The presence of both dopamine- and norepinephrine-synthesizing neurons in the isthmal and medullary tegmentum and in the dorsomedial medulla provides evidence indicating that these regions are homologous to the locus ceruleus, medullary reticular nucleus and area postrema, respectively, in tetrapod brains. In addition, the remarkably dense innervation of TH-IR and DBH-IR fibers and terminals in periventricular regions of the hypothalamus and within the telencephalon suggests that there are potential similarities in the catecholaminergic innervation of forebrain regions of teleost and mammalian brains.

Penetration of immunoreagents in Vibratome-sectioned brain: a light and electron microscopic study.

Immunocytochemical studies on the localization of peptides at the ultrastructural level have most frequently involved the application of the peroxidase--antiperoxidase (PAP) method of immunocytochemistry and the use of the preembedding or postembedding staining procedures. The present study was designed to determine the depth of penetration of Vibratome tissue sections by immunoreagents used in the preembedding method in which immunostaining of unembedded fixed tissue sections is accomplished prior to tissue dehydration and embedment. Our data indicate that penetration of immunoreagents is restricted to the superficial 8-9 micrometers of a 80-micrometers thick Vibratome tissue section of hypothalamus of brain using antisera generated against arginine vasopressin. The final immunoreaction product visualized in a Vibratome tissue section may reflect only a fraction of the amount of hormone contained within the thickness of the tissue section.

Relationship of CRF-immunostained cells and magnocellular neurons in the paraventricular nucleus of rat hypothalamus

The distribution of corticotropin-releasing factor (CRF), vasopressin (VP) and oxytocin (OXY) containing neurons within the magnocellular and parvocellular divisions in the paraventricular nucleus (PVN) of rat hypothalamus is described in brains from normal untreated, colchicine treated and adrenalectomized animals. Double immunostained preparations using glucose oxidase-antiglucose oxidase (GAG) complex combined with PAP complex to visualize two antigens with contrasting colors in the same tissue sections were employed. Separate and distinct populations of cells containing the immunoreactive (ir) elements were seen. Immunostained CRF neurons present in the ventral medial portion of the posterior magnocellular division were juxtaposed to oxytocin-ir perikarya in colchicine treated and adrenalectomized animals. CRF-ir cells were for the most part concentrated in the medial parvocellular component of PVN. An intimate anatomical proximity between CRF-ir and VP-ir perikarya was evident in this medial parvocellular division in brains of adrenalectomized animals; this area is normally VP-ir poor except in the adrenalectomized rats. This extension of VP-ir cells into this CRF rich region and the very close approximation between the two cell bodies suggests potential cell to cell communication following perturbation of the brain-pituitary-adrenal axis. No evidence for the co-existence of two peptidergic systems in the same neuron was apparent in the present study.

ROLE OF THE HYPOTHALAMIC PARAVENTRICULAR NUCLEUS IN CARDIOVASCULAR REGULATION

1. The paraventricular hypothalamic nucleus (PVH) is a complex structure with both neuroendocrine and autonomic functions. It is a major source of vasopressin and the primary source of corticotropin‐releasing factor. In addition, parvicellular PVH neurons have reciprocal connections with brain‐stem autonomic centres and directly innervate sympathetic preganglionic neurons. Evidence is reviewed which indicates that in conscious rats PVH activation increases blood pressure, heart rate, renal nerve activity and plasma renin activity.

Differential Spatial Patterns of Fos Induction Following Generalized Clonic and Generalized Tonic Seizures

The expression of generalized clonic and generalized tonic seizures has been suggested to result from the activation of different and independent neuronal circuits. Using the induction of the c-fos protein (Fos) as a marker of neuronal activity, we identified brain structures that are differentially associated with the expression of electroconvulsive shock-induced generalized clonic and generalized tonic seizures. Expression of either seizure phenotype resulted in a similar bilaterally symmetrical increase in Fos immunoreactivity in many forebrain structures, including the bed nucleus of the stria terminalis, hippocampal dentate gyrus, amygdala, and piriform cortex, compared to controls. However, following tonic hindlimb extension (THE), the degree of labeling in specific thalamic, hypothalamic, and brain stem areas was significantly greater than that of either controls or animals exhibiting clonic seizures. While a greater number of neurons in the hypothalamus (e.g., ventromedial nucleus), subparafascicular thalamic nucleus, peripeduncular area, deep medial superior colliculus, dorsal and lateral central gray, and paralemniscal nuclei were robustly labeled following THE, noticeably fewer cells were immunoreactive following face and forelimb clonic seizure behaviors. These differences were also found to be independent of the stimulus magnitude. In animals stimulated with the same current intensity but expressing either of the two seizure phenotypes, the pattern of Fos induction was consistent with the seizure phenotype expressed. These results demonstrate that specific subsets of neurons are differentially activated following the expression of different generalized seizure behaviors and that activity in discrete mesencephalic and diencephalic structures is more frequently associated with the expression of generalized tonic seizures than with the expression of generalized clonic seizures.

Co-existence of CRF and vasopressin immunoreactivity in parvocellular paraventricular neurons of rat hypothalamus

New dual immunocytochemical staining procedures were used in the same tissue section to elucidate the distribution and co-existence of CRF and vasopressin in parvocellular neuronal perikarya in the paraventricular nucleus (PVN) of rat hypothalamus. CRF immunostained cells were for the most part concentrated in the medial parvocellular component of PVN. Few vasopressin-immunoreactive (ir) neurons were seen in this area in the normal and colchicine-treated animals. Vasopressin-containing neurons predominated in the magnocellular component of PVN. In the adrenalectomized and adrenalectomized-colchicine-treated animals, a dense accumulation of vasopressin-ir cells were observed in the medial parvocellular area of PVN; this region is normally vasopressin-ir poor and CRF-ir rich. The vasopressin immunostained cells appeared to have an anatomical distribution similar to that seen for CRF-containing cell bodies. Results of this study unequivocally establish the co-existence of vasopressin and CRF in the same parvocellular perikarya of PVN following pertubation of the pituitary-adrenal axis.

APOPTOTIC AND NECROTIC CELL DEATH FOLLOWING KINDLING INDUCED SEIZURES

The study was designed to determine which type of cell death occurs following kindling induced seizures, and to determine which neurons die. For this purpose seizures were kindled from the entorhinal cortex. Following a range of 5-85 stage 5 seizures, rats were sacrificed, and the tissue was prepared for analysis. The TUNEL and silver impregnation methods were used to identify apoptotic or necrotic cell death, respectively. These methods were subsequently combined with immunocytochemistry, to determine if diseased neurons expressed somatostatin or the NMDA receptor (NMDAR1). The tissue analysis demonstrated that following kindling induced seizures, 1) hippocampal and extrahippocampal neurons die, 2) some neurons die through apoptosis, others through necrosis, and 3) some of the diseased neurons express somatostatin, others the NMDAR1 and that both subpopulations of neurons are present at hippocampal and extrahippocampal sites.

Increased corticotropin-releasing factor immunoreactivity in select brain sites following kainate-elicited seizures

The literature has focused on the localization, regulation and function of corticotropin-releasing factor (CRF) expressing neurons localized in the paraventricular nucleus (PVN) of hypothalamus. However, less information is available on the expression, regulation, and function of CRF at extrahypothalamic sites. The current study examined the induction of CRF in extrahypothalamic brain sites following generalized clonic seizures induced by kainic acid. At 24 h post seizure onset, there was a marked increase of CRF immunolabeled perikarya in select brain areas, which contained little, if any, CRF in control brains. This CRF-like labeling was observed in olfactory structures such as the main olfactory bulb (internal granular layer), anterior olfactory nucleus, and deep layers of piriform cortex. Other sites of increased CRF-like immunoreactivity included the tenia tecta, inner layers of cingulate cortex, lateral septum, dorsal endopiriform nucleus, fundus striatum, and nucleus of the lateral olfactory tract. Additionally, CRF-like labeling was atypically increased in the amygdala (lateral and basolateral amygdaloid nuclei) and hippocampal formation (pyramidal cells of regions CA1/CA3 and polymorph cells within the dentate hilus). An association between the increased CRF immunoreactivity and neuropathological processes, characteristic of this seizure model, is hypothesized and discussed.

Enkephalin, substance P, and serotonin axonal input to c-fos-like immunoreactive neurons of the rat spinal cord.

Mustard oil, which stimulates small diameter afferents, was used to evoke the expression of the oncogene c-fos in the lumbar spinal cord. C-fos-like immunoreactivity was concentrated in, but not limited to, neuronal nuclei of laminae I and II of the lumbar dorsal horn. Double-label immunocytochemistry was used to determine if neurons which expressed c-fos-like immunoreactivity received axonal input from enkephalin-, substance P- or serotonin-immunoreactive neurons. The analysis of vibratome and semithin plastic-embedded tissue sections demonstrated that the majority of c-fos-like immunoreactive neurons received input from enkephalin-, substance P- or serotonin-immunoreactive axonal varicosities.