T.A. Lovick

The distribution of serotonin, met-enkephalin and β-lipotropin-like immunoreactivity in neuronal perikarya of the cat brainstem

The distribution of methionine-enkephalin, serotonin and beta-lipotropin-containing neurones has been mapped immunohistochemically in the brainstem of the cat after local colchicine injections. Serotonin-containing neurons were found in th raphe in nucleus raph magnus (NRM), nucleus raphe pallidus (NRP) and nucleus raphe obscurus (NRO) and in a band extending cross the ventral reticular formation into nucleus paragigantocellularis lateralis (PGL) and nucleus lateralis reticularis (LRM). Enkephalin-positive cell bodies were also found in these raphe nuclei and in most divisions of the caudal reticular formation. Double labelling studies established that enkephalin and serotonin co-exist within some neurons in NRM and NRP. beta-Lipotropin-positive cell bodies were found principally within NRP and within the ventrolateral reticular formation in PGL and LRM.

GABAergic neurones in the rat periaqueductal grey matter express α4, β1 and δ GABAA receptor subunits: Plasticity of expression during the estrous cycle

Abstract Immunoreactivity for α4, β1 and δ GABAA receptor subunits on neurones in the periaqueductal gray matter was investigated at different stages of the estrous cycle in Wistar rats. Immunostaining for α4, β1 and δ GABAA receptor subunits was present on neurones throughout the periaqueductal gray matter. The numbers of subunit-immunoreactive neurones remained constant during the early phases of the estrous cycle (proestrus to early diestrus) but increased significantly in late diestrus. Dual immunolabeling for the GABA synthesizing enzyme glutamic acid decarboxylase revealed that almost 90% of the subunit-positive cells contained immunoreactivity for glutamic acid decarboxylase. During the early phases of the estrous cycle (proestrus to early diestrus), approximately one third of the glutamic acid decarboxylase-positive population co-localized α4, β1 and δ GABAA receptor subunits. When the number of subunit positive cells increased during late diestrus, the proportion of the glutamic acid decarboxylase-containing population that expressed α4, β1 and δ GABAA receptor subunits almost doubled. We propose that GABAA receptors with the α4β1δ configuration are expressed by GABAergic neurones in the periaqueductal gray matter and that the numbers of cells expressing these subunits are increased in late diestrus in line with falling plasma progesterone levels. Changes in GABAA receptor expression may lead to changes in the excitability of the neural circuitry in the periaqueductal gray matter.

Neurovascular relationships in hippocampal slices: physiological and anatomical studies of mechanisms underlying flow-metabolism coupling in intraparenchymal microvessels.

Experiments were carried out to investigate the functional and anatomical relationships between neuronal elements and cerebral microvessels in 300-350-microm thick coronal hippocampal slices maintained at 33-35 degrees C, obtained from 150-200 g male Wistar rats. Cerebral arterioles (9-22 microm in diameter) were visualized in situ and pre-constricted by 22.0+/-6.6% by the addition of the thromboxane A2 agonist U46619 (75 nM), to the bathing medium. The glutamate agonist N-methyl-D-aspartate (0.01-1 mM) produced a dose-related increase in luminal diameter of pre-constricted vessels. In the presence of 4 microM haemoglobin to scavenge nitric oxide from the extravascular environment of the slice, the increase in diameter evoked by 0.1 mM N-methyl-D-aspartate was significantly reduced from 17.5+/-4.6% to 4.8+/-1.7% indicating that N-methyl-D-aspartate-induced vasodilatation of cerebral microvessels is mediated via a mechanism which involves neuronally-derived nitric oxide. In a parallel anatomical study, beta-nicotinamide adenine dinucleotide phosphate-dependent diaphorase staining was used to reveal the enzyme nitric oxide synthase in vascular endothelium and neurons in slices. A small subpopulation (< 11 cells per slice) of darkly-stained multipolar neurons, 21-32 microm in diameter was observed to give rise to a dense network of fine diaphorase-reactive nerve fibres that ramified throughout the whole of the hippocampus and appeared to come into close apposition with arterioles. Morphometric analysis of the relationship between cerebral microvessels, beta-nicotinamide adenine dinucleotide phosphate, reduced form-dependent diaphorase-reactive neuronal elements and individual pyramidal layer neurons, identified by filling with biocytin, revealed that for a given point on a pyramidal layer neuron, the proximity of the nearest diaphorase-reactive nerve fibre was less than 10 microm, whilst the distance to the nearest arteriole (the smallest functional unit for controlling blood flow) was in excess of 70 microm. Such a distance would probably preclude diffusion of vasoactive metabolites in effective concentrations from the area of increased neuronal activity. We therefore propose that the diaphorase-reactive nerve network constitutes the functional link. It is possible that during periods of increased neuronal activity, spillover of glutamate from synapses may activate the diaphorase-reactive network. Release of nitric oxide from the network in the vicinity of local cerebral arterioles may then produce relaxation of the vascular smooth muscle, enabling increased blood flow into the capillary network supplying the region of increased metabolic activity. This study has shown that the process whereby increases in neuronal activity elicit a local change in cerebral blood flow remains functionally intact in hippocampal slice preparations. Nitric oxide of neuronal origin appears to be involved in mediating the coupling between neurons and cerebral arterioles. Stereological analysis of the relationship between neuronal and vascular elements within hippocampal slices suggested that a small subpopulation of nitric oxide synthase-containing neurons which give rise to a diffuse network of fine nitric oxide synthase-containing nerve fibres that lie in close apposition to cerebral arterioles may provide the anatomical substrate for coupling of blood flow to metabolism.

Co-localization of 5-HT2A-receptor- and GABA-immunoreactivity in neurones in the periaqueductal grey matter of the rat

The dorsal half of the midbrain periaqueductal grey matter (dPAG) functions as a midbrain aversive system. Serotonin exerts anti-aversive effects in the dPAG that are mediated by actions at 5-hydroxytryptamine (5-HT)(1A)- and 5-HT(2A)-receptors. Since at the cellular level, 5-HT(2A)-receptor activation has been shown to evoke excitatory responses in the dPAG, it is possible that anti-aversive 5-HT(2A)-mediated effects are mediated indirectly by activation of inhibitory gamma-aminobutyric acid (GABA)ergic interneurones. In rats, immunoreactivity for 5-HT(2A)-receptors was present on the soma and dendrites of neurones throughout the PAG. Co-localization studies revealed that the majority (>90%) of 5-HT(2A)-receptor-labelled cells also showed immunoreactivity for GABA. These findings may provide an anatomical substrate for 5-HT(2A)-mediated anti-aversive effects in the dPAG.

Bio-imaging of nitric oxide-producing neurones in slices of rat brain using 4,5-diaminofluorescein

4,5-Diaminofluorescein (DAF-2) was used to identify individual nitric oxide (NO)-producing neurones in brain slices in vitro. Coronal slices of midbrain or hippocampus, 300 microm thick from young adult rats, were incubated for 30 min in 1 microM DAF-2 diacetate (DAF-2 DA) and maintained in ACSF at 33 degrees C. Illumination at 450-490 nm revealed punctate fluorescence in neurones in the lateral tegmental nucleus, dorsal raphe nucleus, dorsolateral periaqueductal grey matter, deep collicular layers and cortical areas. Neurones in the hippocampal pyramidal cell layer, molecular layer of the dentate gyrus and the hilus fluoresced also. The fluorescence was abolished by pre-incubation of slices with L-NAME (100 microM-1 mM), the inhibitor of constitutive nitric oxide synthase (NOS), but not by D-NAME (100 microM) or L-NIL (5-50 microM), an inhibitor of inducible NOS. In some superficially located arterioles, there were small regions of bright fluorescence close to the outer smooth muscle wall and diffuse fluorescence within the adjacent smooth muscle cells. A diffuse fluorescence was also seen in some superficially located capillaries. Basal production of NO was not seen within deeper blood vessels. DAF-2 DA offers a sensitive indicator for visualising basal production of NO with high spatial resolution and could provide a means of identifying NOS-containing neurones in brain slices in vitro prior to neurophysiological study.

Projections from brain stem nuclei to the spinal trigeminal nucleus in the cat.

Afferent projections to the trigeminal nucleus oralis and caudalis from the brain stem have been investigated by the use of retrograde transport of horseradish peroxidase in the cat. Both n. oralis and n. caudalis receive a projection from nucleus raphe magnus but not from other raphe nuclei in the medulla or pons. N. oralis and n. caudalis receive a bilateral projection from n. paragigantocellularis lateralis. N. oralis receives a projection from n. reticularis gigantocellularis and n. reticularis parvocellularis but not from n. reticularis magnocellularis. N. caudalis receives only sparse projections from n. reticularis gigantocellularis, n. reticularis parvocellularis and n. reticularis magnocellularis but receives an input from a layer of cells over the pyramids in the rostral medulla, here named n. paramagnocellularis ventralis. The study also revealed the presence of ascending and descending interconnections between n. oralis and n. caudalis, as well as contralateral trigeminal interconnections. Projections from the medial vestibular nuclei, n. praepositus hypoglossi and the facial nucleus to the spinal trigeminal nucleus were also noted. Since the spinal trigeminal nucleus has only sensory functions, the results indicate the source of projections, mainly from raphe and reticular nuclei, which are involved in sensory control in the trigeminal system.

Responses of raphespinal and other bulbar raphe neurones to stimulation of the periaqueductal gray in the cat

The relationship between the periaqueductal gray (PAG) and nucleus raphe magnus (NRM) has been investigated. Electrical stimulation of the PAG in decerebrate or chloralose anaesthetised cats resulted in synaptic activation of raphespinal and other cells in NRM. These results support the view that analgesia of the limbs, trunk and tail produced by stimulation of the PAG in conscious animals may be mediated by synaptic activation of raphespinal neurones.

Involvement of nitric oxide and serotonin in modulation of antinociception and pressor responses evoked by stimulation in the dorsolateral region of the periaqueductal gray matter in the rat.

In rats anaesthetized with alphaxalone/alphadolone, electrical stimulation in the periaqueductal gray matter in the region lying lateral and dorsolateral to the aqueduct produced a pressor response and an increase in the latency of the tail flick response to noxious heat applied to the tail. The antinociception and the pressor response were significantly attenuated following microinjection of 15 nmol 5-hydroxytryptamine at the site of stimulation in the periaqueductal gray matter. Microinjection of an equal volume of 165 mM saline had no effect. The inhibitory effects of 5-hydroxytryptamine were blocked by prior intracerebroventricular administration of 100 microg of the nitric oxide synthase inhibitor L-nitroarginine methyl ester. Neither 5-hydroxytryptamine or L-nitroarginine methyl ester had any effect on resting arterial pressure or on the baseline latency of the tail flick reflex. It is suggested that the inhibitory effects of 5-hydroxytryptamine in the dorsolateral periaqueductal gray matter are normally dependent on the functional integrity of local nitric oxide synthase-containing interneurons. Nitric oxide may act in association with 5-hydroxytryptamine to control the excitability of the aversive system in the midbrain.

Influence of the dorsal and median raphe nuclei on neurons in the periaqueductal gray matter: Role of 5-hydroxytryptamine

In rats anaesthetized with urethane, selective activation of neuronal perikarya in the dorsal raphe nucleus evoked inhibitory (n = 17) and excitatory (n = 10) responses in single neurons recorded in the dorsolateral and lateral sectors of the periaqueductal gray matter and in the adjacent tegmental area. A further 11 cells showed biphasic inhibitory/excitatory responses. Ongoing activity of > 85% of the cells was inhibited by iontophoretic application of 5-hydroxytryptamine (1-70 nA). The duration of the inhibitory response evoked from the dorsal raphe nucleus was increased by 66-75% during iontophoretic application (3-10 nA) of the 5-hydroxytryptamine (serotonin) reuptake blocker paroxetine (five of six cells). In contrast, excitatory responses evoked from the dorsal raphe nucleus were either reduced (n = 3) or replaced by inhibitory responses (n = 2) in the presence of paroxetine. Paroxetine also produced a reduction in baseline firing and potentiated the responses of neurons to iontophoretically applied 5-hydroxytryptamine. Stimulation in the median raphe nucleus did not produce any significant changes in the activity of five neurons tested in the periaqueductal gray matter. It is suggested that the inhibitory influence of the dorsal raphe nucleus on cells in the dorsal half of the periaqueductal gray matter is mediated by 5-hydroxytryptamine. This projection may be involved in modulating the level of excitability of neurons in the midbrain aversive system which integrate defence behaviour. In addition, there appears to be a non-serotonergic excitatory projection from the dorsal raphe nucleus to the periaqueductal gray matter. The functional role of this projection remains obscure.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuronal excitability in the periaqueductal grey matter during the estrous cycle in female wistar rats

Extracellular recordings were made from output neurons in the dorsal half of the periaqueductal gray matter (dPAG) in urethane-anesthetized female Wistar rats. All the neurons were quiescent. A basal level of firing was therefore induced by continuous iontophoretic application of D,L-homocysteic acid (DLH). In the presence of the GABA(A) receptor antagonist bicuculline methiodide (BIC 0-30 nA) the DLH-induced firing increased further, revealing the presence of ongoing GABAergic inhibitory tone on the recorded neurons. The BIC-induced increase in firing rate was significantly greater in neurons recorded during estrus (Est) and late diestrus (LD) compared with proestrus (Pro) and early diestrus (ED) suggesting that GABAergic tone was lower in Est and LD. I.v. injection of the panicogenic cholecystokinin (CCK)(B) receptor agonist pentagastrin (PG, 40 microg kg(-1)) produced an increase in firing rate in 12/17 (70%) of neurons tested in the dPAG. Iontophoretic application of PG (10-30 nA) also produced a current-related increase in firing rate in 73.6% of the neurons tested. The excitatory response was reduced during application of the selective CCK(B) receptor antagonist beta-[2-([2-(8-azaspiro[4.5]dec-8-ylcarbonyl)-4,6-dimethylphenyl]amino)-2-oxoethyl]-(R)-napthalenepropanoic acid (CR2945) (60 nA, n=6). The PG-evoked increase in firing rate was significantly greater in neurons recorded during Est and LD compared with during Pro and ED. Juxtacellular labeling with neurobiotin in eight neurons revealed multipolar cells 12-44 microm diameter with up to six primary dendrites. In three of eight neurons, a filled axon was present and coursed without branching toward the perimeter of the periaqueductal gray matter (PAG). The estrous cycle-related change in responsiveness to BIC and PG suggests that the panic circuitry in the PAG may become more responsive to panicogenic agents during estrus and late diestrus as a consequence of a decrease in the intrinsic level of inhibitory GABAergic tone. The findings may have implications for understanding the neural processes that underlie the development of premenstrual dysphorias in women.