Corticofugal projections to the periaqueductal gray matter in the cat midbrain

Stereotaxic microinjections of horseradish peroxidase (HP) were made into different parts of the rostral and caudal periaqueductal gray (PAG) in cats to study corticofugal projections to the PAG. The method of retrograde axonal transport of HP demonstrated labeled neurons in the I and II somatosensory areas, frontal, cingular and insular cortex of the brain. It was shown that the II somatosensory cortex projects to all the areas of the rostral and caudal PAG. The frontal cortex projects to the dorsolateral quadrant of the PAG. The findings obtained enabled the detection of the morphological substrate of the corticofugal effects on one of the antinociceptive brain structures--the PAG.     

Role of the midbrain periaqueductal gray matter in conditioned reflexes

Unit activity in the midbrain periaqueductal gray matter (PGM) during an instrumental placing reflex, its extinction, differentiation, and conditioned inhibition, was studied in chronic experiments on cats. Spike responses 1–2 sec in duration in 69 (36.7%) of 182 neurons preceded by 400–800 msec the beginning of conditioned-reflex and voluntary intertrial movements. These advanced responses appeared 200 msec before the corresponding advance responses of motor cortical neurons. Fifty-eight neurons (30.9%) responded directly to acoustic stimulation with a latent period of 10–50 msec for 2–6 sec, 19 neurons (10.1%) generated double responses, linked with both the acoustic stimulus and subsequent conditioned-reflex movement, and 42 neurons (22.3%) did not respond to acoustic stimulation, although individual neurons of this group changed the level of their spontaneous activity in response to repeated conditioned stimulation, and this change was maintained for some tens of minutes. Extinction, differentiation, and conditioned inhibition all abolished conditioned-reflex movements, but each type of internal inhibition was accompanied by its own characteristic changes in the firing pattern of PGM neurons. Functional independence of neurons of the first and second groups was demonstrated during extinction and recovery of the conditioned-reflex. The results indicate the important role of PGM not only in the mechanism of the conditioned reflex, but also in the development of its internal inhibition.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 3, pp. 403–419, May–June, 1984.     

Participation of midbrain periaqueductal gray matter in defense conditioning in rats

Responses of neurons of the periaqueductal gray matter (PAG) were studied in chronic experiments on cats during formation and extinction of a defensive conditioned reflex to sound and its differential inhibition. In response to conditioned stimulation these neurons developed phasic-tonic spike responses up to 3 sec in duration. During combination of stimuli these responses were formed long before the conditioned reflex and disappeared long after the latter was extinguished. In the case of an established conditioned reflex, the onset of spike responses occurred 100–200 msec before the appearance of motor responses. An increase in spike activity of tonic character in neurons of PAG preceded voluntary movements by 100–500 msec. The responses of these neurons to presentation of a differential stimulus consisted of groups of spikes 150–200 msec in duration. They were formed with difficulty, and their manifestation was made even more difficult by an interruption during the experiment and by preceding positive stimuli. On the basis of the results a conditioned reflex can be regarded as the result of a multilevel hierarchic process of readjustment of unit activity, which begins in the nonspecific structures of the midbrain.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 15, No. 3, pp. 278–287, May–June, 1983.     

Opioid peptides within the midbrain periaqueductal gray suppress affective defense behavior in the cat

The effects of the methionine-enkephalin analog [D-Ala2-Met5]-enkephalinamide (DAME) upon the threshold for affective defense behavior were determined following microinjections placed into midbrain periaqueductal gray sites from which this response was elicited. Affective defense behavior was elicited by electrical stimulation through a cannula electrode situated in the dorsal aspect of the midbrain periaqueductal gray. Dose-response curves characterizing the effects of DAME upon affective defense behavior were determined utilizing the following doses: 0.25, 0.5 and 1.0 microgram in 0.5 microliter saline, pH = 7.4 or vehicle control (saline). Response thresholds were tested 10-30, 30-60, 60-90, 120-150, 180-210, 1440-1470 and 2880-2910 min postinjection. The results obtained indicated that injections of DAME at a dose of 1.0 microgram/0.5 microliter produced significant, long duration elevations in affective defense thresholds, lasting up to 1440-1470 min postinjection. Lower doses of DAME (0.25 and 0.5 microgram/0.5 microliter) also resulted in significant increases in affective defense thresholds, but these effects were of shorter durations (60-90 and 120-150 min) postinjection, respectively. The suppressive effects of DAME were blocked when animals were pretreated with naloxone (10 micrograms/0.5 microliter) microinjected into the same midbrain periaqueductal gray site into which 0.25 microgram DAME was injected and affective defense behavior was elicited.     

Intranuclear bodies in neurons of the periaqueductal gray matter in the cat.

The nucleoplasm of neurons in the nucleus lateralis of the periqueductal gray matter in the cat contains fibrillar structures which have no limiting membranes. These intranuclear bodies are associated with neither the nucleolus nor the nuclear membrane and have two characteristic forms. The first, the rodlet, is a compact bundle of fibrils 2 to 8 nm in diameter. It is usually elongated in shape although it appears spherical when sectioned transversely. This rod-like structure appears to correspond to Roncoroni's rodlet or the accessory body of Cajal in light microscopy. The second and more commonly observed form is a long slender bundle of five rows of parallel fibrils. Although similar intranuclear structures have frequently been observed in the highly differentiated neurons of the sympathetic ganglia and the retina, this is the first report of their pbesence in the undifferentiated neurons of the isodendritic core of the brainstem.     

Cytoarchitecture of the periaqueductal gray matter in the cat: a quantitative Nissl study

We studied the periaqueductal gray matter (PAG) in cresyl-violet-stained serial sections from 5 cats applying quantitative determinations. No significant variations were observed in the cytological aspects in the various sites examined (lateral, ventral and dorsal regions, and external and internal portions). The neuronal density was constant in the different regions, but showed a gradual and significant increase in the most external regions of the PAG. Our findings do not, therefore, confirm the existence in the PAG of subnuclei with a specific cytoarchitecture; this does not, however, rule out the possibility that there are specific regions for connections, histochemical properties or functions.     

Modulation of neuron activity of the midbrain periaqueductal gray matter influenced by monoaminergic brainstem structures

A study was done on base activity and changes in base activity (BA) of neurons in periaqueductal gray matter (PAG) during stimulation of monoaminergic structures of the brainstem: the nucleus raphe magnus (NRM), the locus coeruleus (LC), and the substantia nigra (SN), in rats anesthetized with hexenal (200 mg/kg). Three types of PAG neurons that differed in BA structure were identified. NRM, LC and SN stimulation changed BA only in type III neurons. Stimulation of these structures evoked an increase in BA frequency in 11.0–14.5%, and inhibition in 31.0–47.5% of type III neurons. Simultaneous stimulation of two structures led to a marked drop in intensity of effects. A depressing effect on BA was always detected if stimulation of one of the structures suppressed BA. Stimulation of two structures, with one being the NRM, was most effective. The role of PAG in the organization of the brain-stem component of the antinociceptive mechanism is discussed.A. A. Bogomolets Institute of Physiology, Ukrainian Academy of Sciences, Kiev. Translated from Neirofiziologiya, Vol. 24, No. 1, pp. 52–60, January–February, 1992.     

c-Fos expression in the midbrain periaqueductal gray during static muscle contraction

The periaqueductal gray (PAG) of the midbrain is involved in the autonomic regulation of the cardiovascular system. The purpose of this study was to determine if static contraction of the skeletal muscle, which increases arterial blood pressure and heart rate, activates neuronal cells in the PAG by examining Fos-like immunoreactivity (FLI). Muscle contraction was induced by electrical stimulation of the L7 and S1 ventral roots of the spinal cord in anesthetized cats. An intravenous infusion of phenylephrine (PE) was used to selectively activate arterial baroreceptors. Extensive FLI was observed within the ventromedial region (VM) of the rostral PAG, the dorsolateral (DL), lateral (L), and ventrolateral (VL) regions of the middle and caudal PAG in barointact animals with muscle contractions, and in barointact animals with PE infusion. However, muscle contraction caused a lesser number of FLI in the VM region of the rostral PAG, the DL, L, and VL regions of the middle PAG and the L and VL regions of the caudal PAG after barodenervation compared with barointact animals. Additionally, the number of FLI in the DL and L regions of the middle PAG was greater in barodenervated animals with muscle contraction than in barodenervated control animals. Thus these results indicated that both muscle receptor and baroreceptor afferent inputs activate neuronal cells in regions of the PAG during muscle contraction. Furthermore, afferents from skeletal muscle activate neurons in specific regions of the PAG independent of arterial baroreceptor input. Therefore, neuronal cells in the PAG may play a role in determining the cardiovascular responses during the exercise pressor reflex.     

c-Fos expression in the midbrain periaqueductal gray after chemoreceptor and baroreceptor activation

The pattern of Fos-like immunoreactivity (FLI) in the periaqueductal gray (PAG) associated with activation of arterial chemoreceptors versus baroreceptor afferents was examined in urethane-anesthetized rats. Chemoreflex responses elicited by repeat intravenous injections of potassium cyanide (KCN; 90 microg/kg) significantly increased FLI in all columns of the PAG relative to saline-injected animals. Pressor responses elicited by intravenous phenylephrine (PE) produced a similar pattern of increased FLI throughout the PAG except in the dorsomedial and lateral columns of the caudal PAG, where FLI was minimal. Chemoreflex responses were unaltered by blockade of excitatory amino acid receptors in the dorsomedial PAG, and < 10% of the neurons of the caudal PAG that expressed FLI after KCN stimulation were retrogradely labeled from the A5 region of the caudal ventrolateral pons. These results indicate that integration of chemoreceptor inputs occurs primarily in the dorsal and lateral columns of the caudal PAG, but these neurons have little direct descending influence over lower brain stem regions integral to the central arterial chemoreflex arc.     

Effects of stimulating the midbrain central gray matter on neuronal response in the cat ventroposteromedial thalamic nucleus

The effects of stimulating the midbrain central gray matter (CGM) on neuronal response in the ventroposteromedial (VPM) nucleus produced by stimulating tooth pulp, A-alpha and A-delta fibers of the intraorbital nerve and the caudal nucleus of the spinal trigeminal tract (CN STT) were investigated during experiments on cats under thiopental-chloralose anesthesia. It was found that applying trains of stimuli to the CGM produced excitatory responses in a proportion of the test neurons with latencies of up to 30 msec. Application of conditioning stimulus to the CGM led to suppression of response of efferent stimulation in neurons belonging to low-threshold, convergent, and high-threshold groups. Responses produced in 40% of neurons by stimulating tooth pulp and A-delta fibers of the suborbital nerve, as well as those evoked in 26.4% of thalamic VPM cells by stimulating A-alpha fibers of the suborbital nerve were completely suppressed. The inhibitory effect found when stimulating CGM on response in certain neurons, produced by stimulating both the peripheral nerve and the CN STT, would indicate that the CGM could exert an influence on the activity of thalamic VPM neurons directly.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 5, pp. 688–694, September–October, 1988.     

GABA-immunoreactive neurons and terminals in the cat periaqueductal gray matter: A light and electron microscopic study

Immunocytochemical and electron microscopic methods were used to study the GABAergic innervation in adult cat periaqueductal gray matter (PAG). A mouse monoclonal antibody against γ -aminobutyric acid (GABA) was used to visualize the inhibitory neuronal system of PAG. At light microscopy, GABA-immunopositive (GABA_IP) neurons formed two longitudinally oriented columns in the dorsolateral and ventrolateral PAG that accounted for 36% of the neuronal population of both PAG columns; their perikaryal cross-sectional area was smaller than that of unlabeled (UNL) neurons found in the same PAG subdivisions. At electron microscopic level, patches of GABA immunoreactivity were readily detected in neuronal cell bodies, proximal and distal dendrites, axons and axon terminals. Approximately 35–36% of all terminals were GABA_IP; they established symmetric synapses with dendrites (84.72% of the sample in the dorsolateral PAG and 86.09% of the sample in the ventrolateral PAG) or with cell bodies (7–10% of the sample). Moreover, 49.15% of GABA_IP axon terminals in the dorsolateral and 52.16% in the ventrolateral PAG established symmetric synapses with GABA_IP dendrites. Immunopositive axon terminals and unlabeled terminals were also involved in the formation of a complex synaptic arrangment, i.e. clusters of synaptic terminals in close contact between them that were often observed in the PAG neuropil. Moreover, a fair number of axo-axonic synapses between GABA_IP and/or UNL axon terminals were present in both PAG subdivisions. Several dendro-dendritic synapses between labeled and unlabeled dendrites were also observed in both PAG subdivisions. These results suggest that in the cat PAG there exist at least two classes of GABArgic neurons. The first class could exert a tonic control on PAG projecting neurons, the second could act on those GABAergic neurons that in turn keep PAG projecting neurons under tonic inhibition. The functional implications of this type of GABAergic synapse organization are discussed in relation to the dishinibitory processes that take place in the PAG.     

Activation of the midbrain periaqueductal gray induces airway smooth muscle relaxation.

In this study, we examined effects of chemical stimulation of the ventrolateral region of the midbrain periaqueductal gray (vl PAG) on airway smooth muscle tone. We observed that in anesthetized, paralyzed, and artificially ventilated ferrets, vl PAG stimulation elicited airway smooth muscle relaxation. To clarify the mechanisms underlying this observation, we examined the GABA-GABA(A) receptor signaling pathway by 1) examining the expression of GABA(A) receptors on airway-related vagal preganglionic neurons (AVPNs) located in the rostral nucleus ambiguus region (rNA), by use of receptor immunochemistry and confocal microscopy; 2) measuring GABA release within the rNA by using microdialysis; and 3) performing physiological experiments to determine the effects of selective blockade of GABA(A) receptors expressed by AVPNs in the rNA region on vl PAG-induced airway relaxation, thereby defining the role of the GABA(A) receptor subtype in this process. We observed that AVPNs located in the rNA region do express the GABA(A) receptor beta-subtype. In addition, we demonstrated that activation of vl PAG induced GABA release within the rNA region, and this release was associated with airway smooth muscle relaxation. Blockade of the GABA(A) receptor subtype expressed by AVPNs in the rNA by bicuculline diminished the inhibitory effects of vl PAG stimulation on airway smooth muscle tone. These data indicate, for the first time, that activation of vl PAG dilates the airways by a release of GABA and activation of GABA(A) receptors expressed by AVPNs.     

Micturition-suppressing region in the periaqueductal gray of the mesencephalon of the cat

The periaqueductal gray (PAG) of the mesencephalon has been implicated to be involved in the control of micturition. We investigated the micturition-suppressing region in the PAG of the cat. Decerebrated 27 adult cats were used. A microelectrode was inserted stereotaxically into the PAG, and a region was searched where electrical stimulation suppressed isovolumetric bladder contractions. Simultaneous stimulation of the pontine micturition center (PMC) and micturition-suppressing region in the PAG was performed before and after an injection of bicuculline (GABA(A) blocker) into the PMC. The micturition-suppressing region was found at the dorsolateral margin of the rostral PAG. Bladder contractions were not provoked by simultaneous stimulation of the PMC and micturition-suppressing region in the PAG. However, after bicuculline injection into the PMC, partial bladder contractions were provoked by simultaneous stimulation of the PMC and the micturition-suppressing region in the PAG. These results suggest that the dorsolateral margin of the rostral PAG includes the micturition-suppressing region that seems to have neural connections with the PMC. GABA is assumed to be one of the neurotransmitters that are involved in the PMC inhibition from the micturition-suppressing region in the PAG.     

G protein activation by endomorphins in the mouse periaqueductal gray matter

The midbrain periaqueductal gray matter (PAG) is an important brain region for the coordination of mu-opioid-induced pharmacological actions. The present study was designed to determine whether newly isolated mu-opioid peptide endomorphins can activate G proteins through mu-opioid receptors in the PAG by monitoring the binding to membranes of the non-hydrolyzable analog of GTP, guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS). An autoradiographic [(35)S]GTPgammaS binding study showed that both endomorphin-1 and -2 produced similar anatomical distributions of activated G proteins in the mouse midbrain region. In the mouse PAG, endomorphin-1 and -2 at concentrations from 0.001 to 10 microM increased [(35)S]GTPgammaS binding in a concentration-dependent manner and reached a maximal stimulation of 74.6+/-3.8 and 72.3+/-4.0%, respectively, at 10 microM. In contrast, the synthetic selective mu-opioid receptor agonist [D-Ala(2),NHPhe(4), Gly-ol]enkephalin (DAMGO) had a much greater efficacy and produced a 112.6+/-5.1% increase of the maximal stimulation. The receptor specificity of endomorphin-stimulated [(35)S]GTPgammaS binding was verified by coincubating membranes with endomorphins in the presence of specific mu-, delta- or kappa-opioid receptor antagonists. Coincubation with selective mu-opioid receptor antagonists beta-funaltrexamine or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH(2) (CTOP) blocked both endomorphin-1 and-2-stimulated [(35)S]GTPgammaS binding. In contrast, neither delta- nor kappa-opioid receptor antagonist had any effect on the [(35)S]GTPgammaS binding stimulated by either endomorphin-1 or -2. These findings indicate that both endomorphin-1 and -2 increase [(35)S]GTPgammaS binding by selectively stimulating mu-opioid receptors with intrinsic activity less than that of DAMGO and suggest that these new endogenous ligands might be partial agonists for mu-opioid receptors in the mouse PAG.     

Effects of lordosis-relevant neuropeptides on midbrain periaqueductal gray neuronal activity in vitro.

Certain neuropeptides can facilitate lordosis by acting on midbrain periaqueductal gray (PAG) in estrogen-primed female rats. Here, we investigated responses of individual PAG neurons in vitro, to five neuropeptides: substance P (SP), luteinizing hormone-releasing hormone (LHRH), prolactin (PRL), oxytocin (OT), and thyrotropin-releasing hormone (TRH). Substance P, OT, and TRH excited spontaneous activity of PAG neurons through neurotransmitter-like actions in a dose-dependent manner, whereas LHRH and PRL virtually never affected PAG neurons this way. Oxytocin acted through oxytocin receptors located on the recorded PAG neurons, since excitatory actions of OT were 1) not abolished by synaptic blockade, 2) mimicked by the OT-specific agonist [Thr4, Gly7]OT but not by arginine vasopressin, and 3) blocked by the OT-specific antagonist [d(CH2)5,Tyr(Me)2,Orn8]vasotocin. Although LHRH had no neurotransmitter-like action on spontaneous activity of PAG neurons, it, as well as SP, could modulate responses of some dorsal PAG neurons to GABAA and GABAB agonists or norepinephrine. Neuromodulatory actions of LHRH and SP could help facilitate lordosis through PAG neurons.