Stereological analysis of the hypothalamic hypocretin/orexin neurons in an animal model of depression.

Affective disorders often occur in combination with disrupted sleep-wake cycles and abnormal fluctuations in hypothalamic neurotransmitters. Hypocretin (orexin) is a hypothalamic neuropeptide linked to narcolepsy, a sleep-related disorder characterized by profound disturbances in the normal sleeping pattern and variable degrees of depression. Wistar-Kyoto (WKY) rats exhibit depressive characteristics and patterns of sleep disruption similar to that observed in depressed human patients. In this study we sought to determine whether the total number or the size of hypothalamic hypocretin neurons in WKY rats differ from their control, Wistar (WIS) rats. Immunocytochemical and stereological methods were applied to quantify hypocretin-1 containing neurons in the hypothalamus. The study revealed 18% fewer hypocretin-1 positive neurons as well as a 15% decrease in average neuronal soma size of hypocretin-1 producing cells in the hypothalamus of WKY rats compared to WIS rats. These findings support the view that reduced number or size of hypothalamic hypocretinergic neurons may underlie the disrupted sleep pattern associated with depressive characteristics in WKY rats.

Expression of alpha-7 nAChRs on spinal cord-brainstem neurons controlling inspiratory drive to the diaphragm.

In the present study, we determined whether alpha-7 subunit containing nicotinic acetylcholine receptors (nAChRs) are expressed by neurons within the pre-Botzinger complex (pre-BotC), bulbospinal, and phrenic motor nuclei in the rat. alpha-7 Immunohistochemistry combined with cholera toxin B (CTB), a retrograde tracer was used to detect expression of alpha-7 nAChRs by phrenic motor and bulbospinal neurons. Neurokinin-1 receptor immunoreactivity was used as a marker for pre-BotC neurons. Of the CTB-positive neurons in the phrenic nuclei, 60% exhibited immunoreactivity for alpha-7 nAChRs. Of the bulbospinal neurons in the paramedian reticular nuclei (PMn), gigantocellular nuclei (Gi), raphe nuclei, rostral ventrolateral medulla (RVLM) and nucleus tractus solitarius, 20-50% were found to express alpha-7 nAChR immunoreactivity. Of the peudorabies virus (PRV) labeled bulbospinal neurons in PMn, Gi, raphe and RVLM, 9-12% co-expressed alpha-7 nAChRs. Immunoreactivity for alpha-7 nAChRs was also detected in 57% of the neurokinin-1 receptor containing neurons presumed to reside in pre-BotC. These findings suggest that nicotinic cholinergic regulation of the chest wall pumping muscles may occur at multiple levels of the central nervous system.

Paraventricular vasopressin-containing neurons project to brain stem and spinal cord respiratory-related sites.

We studied in the rat projections of vasopressin-containing neurons of the paraventricular nucleus (PVN) to phrenic nuclei and to the pre-Botzinger complex (pre-BotC). In addition, we determined vasopressin receptor expression within the pre-BotC and the physiological effects of vasopressin on respiratory drive and arterial blood pressure when injected into the pre-BotC. Retrograde tracing with cholera toxin B subunit (CT-b) showed that a subpopulation of vasopressin-containing PVN neurons project to phrenic nuclei and the pre-BotC. The latter region, identified by expression of neurokinin-1 receptors, contained a subpopulation of neurons that were immunoreactive for the vasopressin type 1 receptor (V(1)R). Microinjection of vasopressin in the pre-BotC (0.2 nmol/200 nl) significantly increased diaphragm electromyographic activity and frequency discharge (P<0.05). In addition, vasopressin increased blood pressure and heart rate (P<0.05). These data indicate that PVN vasopressin-containing neurons innervate respiratory-related regions of the medulla oblongata and spinal cord and when vasopressin is released at these sites, it may increase respiratory drive via activation of the distinct V(1)R.

CO(2)-induced c-Fos expression in hypothalamic vasopressin containing neurons.

Following exposure of anesthetized and unanesthetized rats to hypercapnic stress, arginine vasopressin (AVP)-containing neurons of the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei were examined for expression of the c-fos gene encoded protein (c-Fos). In addition, we determined whether AVP-containing PVN neurons activated by hypercapnia project to phrenic nuclei. In adult control rats, only scant c-Fos-like immunoreactive neurons were observed within the hypothalamic nuclei. A marked increase in c-Fos positive cells was induced after 2 h of breathing a gas mixture with elevated CO(2) (5% CO(2), 21% O(2) and 74% N(2), or 1 h following breathing of 12% CO(2,) 21% O(2,) and 67% N(2)). Colocalization studies of AVP and c-Fos protein revealed that in the PVN, 75% of AVP-containing cells expressed c-Fos immunoreactivity. c-Fos and AVP were coexpressed in 60% of SON neurons in anesthetized rats. In addition, retrograde labeling studies with cholera toxin b subunit (CTb) revealed that a subpopulation of PVN cells (15%) that project to phrenic nuclei are activated by hypercapnia, as indicated by c-Fos expression. These results indicate that (i) PVN and SON AVP-containing neurons are part of the neuronal networks that react to hypercapnic exposure; and (ii) a subset of CO(2) reactive PVN cells innervate phrenic nuclei.

Cocaine and ventral brainstem neurotoxicity

One of the complications of cocaine abuse is central cardiorespiratory failure. However, the site and mechanism of cocaine are unknown. The present study evaluates the effect of cocaine applied topically to the caudal and intermediate areas on the ventrolateral surface of the brainstem. These areas are known to be involved in the CO2 /pH chemosensory drive to respiration and in vasomotor control. Cats were anesthetized with urethane (2.0 g/kg), the trachea cannulated and the ventro-lateral surface of medulla oblongata (VMS) exposed. Cocaine prepared in mock CSF pH 7.4 was applied bilaterally to chemosensitive zones using pledgets. The effect of procaine was also tested. Tidal volume (Vt), respiratory frequency (f), arterial blood pressure (BP) and heart rate (HR) were monitored. Cocaine (62.5 ug/site) produced a significant decrease in minute ventilation (Ve) and blood pressure (BP) (p<0.05); the cuadal area was more sentive. In equimolar doses to cocaine, procaine (50 ug/site) produced small but significant effects on BP with no changes in Ve however, twice the equimolar dose(100 ug/site), produced respiratory responses similar to that of cocaine. Alpha and beta adrenoceptor antagonists prazosin (10 ug.site) and propranolol (16.7 ug/site) respectively, failed to alter the hypotensive or respiratory depressant effect of cocaine. Only animals that were hypotensive before or during physostigmine pretreatment (5 ug.site) experienced cardiorespiratory failure upon administration of cocaine. Carbachol (2.5 ug/site) had no effect on the cocaine induced cardiorespiratory responses. The present data suggest that (1) central cocaine neurotoxicity may result from interaction of cocaine with VMS sites; (2) the mechanism of action of cocaine at these sites is similar to that of procaine and does not appear to involve adrenergic receptors; and (3) pretreatment with the involve adrenergic receptors; and (3) pretreatment with the cholinomimetic physostigmine was effective in protecting animals from cocaine induced respiratory failure, its efficacy being limited to those animals that were not hypotensive during pretreatment. (AU)

Sexual dimorphism in the basilar part of the occipital bone of the dog (canis familiaris)

Sexual dimorphism in the surface markings of the basilar part of the occipital bone of 34 male and 46 female dog's skulls were studied with the following results: 1 of 34 male skulls showed a female type surface marking, 4 of the 46 female skulls showed surface markings of the opposite sex, while 2 of the male and 3 of the female skulls could not be classified as belonging to either type, so that 10 out of the 80 skulls could not be identified correctly, indicating that 87.5 percent of the skulls could be identified positively. Considering that there are hardly any sexual differences in the other surface markings or in the dentition of the dog's skull, these findings may be of some help in sexing the skull of the dog (AU)

Analysis of respiratory response patterns following electrical stimulation of the medulla oblongata - abstract

The exact location of the "Respiratory Centre" and its possible demarcation into anatomically distinct "inspiratory" and expiratory half centres" have remained the subject of much controversy. In this study the medulla oblongata of 105 cats (1.65-3.40 kg, choralose-urethane anaesthesia) was exposed from the ventral surface, and the effect of the submaximal stimulation (40/sec., 1m sec., 1-4V) on respiration was investigated. The respiratory response pattern was analysed, and group according to the qualitative characteristics into (a) Inspiratory (4 types), (b) expiratory (two types) and (c) Mixed (3 types) effects. The observed response types were then charted on the medullary sections at the points where they were elicited. Taken together, inspiratory responses in the medullary cros sections are seen to be concentrated about three zones: (a) 10mm rostral (pontomedullary border) to the Obex level, (b) from the Obex level up to 4mm rostral of it, and (c) 4-9 mm caudal from the Obex. Expiratory reactions may be elicited in a region extending from 8 mm rostral to 2 mm caudal from the Obex. Expiratory effects were constantly 4-6mm rostral to the Obex on the ventrolateral aspect, even at the surface. Inspiratory and expiratory zones are in close contact forming together a rostrocaudally orientated semispiral, suggesting perhaps a functional interaction. The "respiratory centre" (defined here as structures, the stimulation of which results in respiratory arrest in the inspiratory or expiratory positions) may be demarcated into "inspiratory" and "expiratory" half centres, in agreement with Pitts et al. (Am. J. Physiol. 126, 673-688, 1939). It is located laterally in the formatio reticularis at the Obex level. At the transition between these two half centres there is a high degree of overlapping, explaining thereby the findings of Liljestrand (Acta. Physiol Scand. 29 suppl. 106, 321-393, 1953) of an intermingling pool of inpiratory and expiratory neurones. Mixed effects showing an increase in amplitude involving both inspiration and expiration were found concentrated ventrally in both rostral and caudal superficial chemosensitive zones (J. Appl. Physiol. 18, 523-533, 1963 and Pflugers Arch. 312, 189-205, 1969). Extending caudally from the caudal chemosensitive zone, the response is characterized by an inspiratory shift from the end-expiratory position. Ablation studies indicate that this type of response is representative, not of intrinsic respiratory centre activity, but of efferent pathway activity from the respiratory centre (AU)