Role of the dorsal periaqueductal gray in posttraumatic stress disorder: mediation by dopamine and neurokinin.

In susceptible individuals, exposure to intensely traumatic life events can lead to the development of posttraumatic stress disorder (PTSD), including long-term dysregulation of the contextual processing of aversive stimuli, the overgeneralization of learned fear, and impairments in the ability to learn or respond to safety signals. The neuropathophysiological changes that underlie PTSD remain incompletely understood. Attention has focused on forebrain structures associated with fear processing. Here we consider evidence from human and animal studies that long-lasting changes in functional connectivity between the midbrain periaqueductal gray (dPAG) and amygdala may be one of the precipitating events that contribute to PTSD. Long-lasting neuroplastic changes in the dPAG can persist after a single aversive stimulation and are pharmacologically labile. The early stage (at least up to 24 h post-stimulation) involves neurokinin-1 receptor-mediated events in the PAG and amygdala and is also regulated by dopamine, both of which are mainly involved in transferring ascending aversive information from the dPAG to higher brain structures, mainly the amygdala. Changes in the functional connectivity within the dPAG-amygdala circuit have been reported in PTSD patients. We suggest that further investigations of plasticity and pharmacology of the PAG-amygdala network provide a promising target for understanding pathophysiological circuitry that underlies PTSD in humans and that dopaminergic and neurokininergic drugs may have a potential for the treatment of psychiatric disorders that are associated with a dysfunctional dPAG.

Chronic implantation of cuff electrodes on the pelvic nerve in rats is well tolerated and does not compromise afferent or efferent fibre functionality.

OBJECTIVE: Neuromodulation of autonomic nerve activity to regulate physiological processes is an emerging field. Vagal stimulation has received most attention whereas the potential of modulate visceral function by targeting autonomic nerves within the abdominal cavity remains under-exploited. Surgery to locate intra-abdominal targets is inherently more stressful than for peripheral nerves. Electrode leads risk becoming entrapped by intestines and loss of functionality in the nerve-target organ connection could result from electrode migration or twisting. Since nociceptor afferents are intermingled with similar-sized visceral autonomic fibres, stimulation may induce pain. In anaesthetised rats high frequency stimulation of the pelvic nerve can suppress urinary voiding but it is not known how conscious animals would react to this procedure. Our objective therefore was to determine how rats tolerated chronic implantation of cuff electrodes on the pelvic nerve, whether nerve stimulation would be aversive and whether nerve-bladder functionality would be compromised. APPROACH: We carried out a preliminary de-risking study to investigate how conscious rats tolerated chronic implantation of electrodes on the pelvic nerve, their responsiveness to intermittent high frequency stimulation and whether functionality of the nerve-bladder connection became compromised. MAIN RESULTS: Implantation of cuff electrodes was well-tolerated. The normal diurnal pattern of urinary voiding was not disrupted. Pelvic nerve stimulation (up to 4 mA, 3 kHz) for 30 min periods evoked mild alerting at stimulus onset but no signs of pain. Stimulation evoked a modest (<0.5 °C) increase in nerve temperature but the functional integrity of the nerve-bladder connection, reflected by contraction of the detrusor muscle in response to 10 Hz nerve stimulation, was not compromised. SIGNIFICANCE: Chronic implantation of cuff electrodes on the pelvic nerve was found to be a well-tolerated procedure in rats and high frequency stimulation did not lead to loss of nerve functionality. Pelvic nerve stimulation has development potential for normalizing voiding dysfunction in conscious rats.

Effect of electrical vs. chemical deep brain stimulation at midbrain sites on micturition in anaesthetized rats.

AIM: To understand how deep brain stimulation of the midbrain influences control of the urinary bladder. METHODS: In urethane-anaesthetized male rats, saline was infused continuously into the bladder to evoke cycles of filling and voiding. The effect of electrical (0.1-2.0 ms pulses, 5-180 Hz, 0.5-2.5 V) compared to chemical stimulation (microinjection of D,L-homocysteic acid, 50 nL 0.1 M solution) at the same midbrain sites was tested. RESULTS: Electrical stimulation of the periaqueductal grey matter and surrounding midbrain disrupted normal coordinated voiding activity in detrusor and sphincters muscles and suppressed urine output. The effect occurred within seconds was reversible and not secondary to cardiorespiratory changes. Bladder compliance remained unchanged. Chemical stimulation over the same area using microinjection of D,L-homocysteic acid (DLH) to preferentially activate somatodendritic receptors decreased the frequency of micturition but did not disrupt the coordinated pattern of voiding. In contrast, chemical stimulation within the caudal ventrolateral periaqueductal grey, in the area where critical synapses in the micturition reflex pathway are located, increased the frequency of micturition. CONCLUSION: Electrical deep brain stimulation within the midbrain can inhibit reflex micturition. We suggest that the applied stimulus entrained activity in the neural circuitry locally, thereby imposing an unphysiological pattern of activity. In a way similar to the use of electrical signals to 'jam' radio transmission, this may prevent a synchronized pattern of efferent activity being transmitted to the spinal outflows to orchestrate a coordinated voiding response. Further experiments to record neuronal firing in the midbrain during the deep brain stimulation will be necessary to test this hypothesis.

Fluoxetine elevates allopregnanolone in female rat brain but inhibits a steroid microsomal dehydrogenase rather than activating an aldo-keto reductase.

BACKGROUND AND PURPOSE: Fluoxetine, a selective serotonin reuptake inhibitor, elevates brain concentrations of the neuroactive progesterone metabolite allopregnanolone, an effect suggested to underlie its use in the treatment of premenstrual dysphoria. One report showed fluoxetine to activate the aldo-keto reductase (AKR) component of 3α-hydroxysteroid dehydrogenase (3α-HSD), which catalyses production of allopregnanolone from 5α-dihydroprogesterone. However, this action was not observed by others. The present study sought to clarify the site of action for fluoxetine in elevating brain allopregnanolone. EXPERIMENTAL APPROACH: Adult male rats and female rats in dioestrus were treated with fluoxetine and their brains assayed for allopregnanolone and its precursors, progesterone and 5α-dihydroprogesterone. Subcellular fractions of rat brain were also used to investigate the actions of fluoxetine on 3α-HSD activity in both the reductive direction, producing allopregnanolone from 5α-dihydroprogesterone, and the reverse oxidative direction. Fluoxetine was also tested on these recombinant enzyme activities expressed in HEK cells. KEY RESULTS: Short-term treatment with fluoxetine increased brain allopregnanolone concentrations in female, but not male, rats. Enzyme assays on native rat brain fractions and on activities expressed in HEK cells showed fluoxetine did not affect the AKR producing allopregnanolone from 5α-dihydroprogesterone but did inhibit the microsomal dehydrogenase oxidizing allopregnanolone to 5α-dihydroprogesterone. CONCLUSIONS AND IMPLICATIONS: Fluoxetine elevated allopregnanolone in female rat brain by inhibiting its oxidation to 5α-dihydroprogesterone by a microsomal dehydrogenase. This is a novel site of action for fluoxetine, with implications for the development of new agents and/or dosing regimens to raise brain allopregnanolone.

Estrous cycle and stress: influence of progesterone on the female brain

The female brain operates in a constantly changing chemical milieu caused by cyclical changes in gonadal hormones during the estrous cycle (menstrual cycle in women). Such hormones are highly lipophilic and pass readily from the plasma to the brain where they can influence neuronal function. It is becoming clear that the rapid reduction in peripheral circulating progesterone, which occurs during the late diestrous phase of the cycle, can trigger a withdrawal-like response, in which changes in GABA A receptor expression render hyper-responsive certain brain areas involved in processing responses to stressful stimuli. The periaqueductal gray matter (PAG) is recognised as an important region for integrating anxiety/defence responses. Withdrawal from progesterone, via actions of its neuroactive metabolite allopregnanolone, triggers up-regulation of extrasynaptic GABA A receptors on GABAergic neurons in the PAG. As a consequence, ongoing GABAergic tone on the output cells decreases, leading to an increase in functional excitability of the circuitry and enhanced responsiveness to stressful stimuli during the late diestrous phase. These changes during late diestrus could be prevented by short-term neurosteroid administration, timed to produce a more gradual fall in the peripheral concentration of allopregnanolone than the rapid decrease that occurs naturally, thus removing the trigger for the central withdrawal response.

Estrous cycle and stress: influence of progesterone on the female brain.

The female brain operates in a constantly changing chemical milieu caused by cyclical changes in gonadal hormones during the estrous cycle (menstrual cycle in women). Such hormones are highly lipophilic and pass readily from the plasma to the brain where they can influence neuronal function. It is becoming clear that the rapid reduction in peripheral circulating progesterone, which occurs during the late diestrous phase of the cycle, can trigger a withdrawal-like response, in which changes in GABA(A) receptor expression render hyper-responsive certain brain areas involved in processing responses to stressful stimuli. The periaqueductal gray matter (PAG) is recognised as an important region for integrating anxiety/defence responses. Withdrawal from progesterone, via actions of its neuroactive metabolite allopregnanolone, triggers up-regulation of extrasynaptic GABA(A) receptors on GABAergic neurons in the PAG. As a consequence, ongoing GABAergic tone on the output cells decreases, leading to an increase in functional excitability of the circuitry and enhanced responsiveness to stressful stimuli during the late diestrous phase. These changes during late diestrus could be prevented by short-term neurosteroid administration, timed to produce a more gradual fall in the peripheral concentration of allopregnanolone than the rapid decrease that occurs naturally, thus removing the trigger for the central withdrawal response.

GABAergic control of micturition within the periaqueductal grey matter of the male rat.

In urethane-anaesthetised rats continuous infusion of saline into the bladder (6 ml h⁻¹) evoked periodic sharp rises in intravesicular pressure accompanied by rhythmic bursting of external urethral sphincter (EUS) EMG and expulsion of urine from the urethral meatus. Microinjection of the GABA agonist muscimol (250 pmol) into the caudal ventrolateral periaqueductal grey (PAG), but not at other sites in the PAG, either depressed reflex voiding frequency (-60%, n = 7) and tonic EUS EMG activity (-38%, n = 6) or completely inhibited voiding (four sites). Microinjection of the GABA antagonist bicuculline (BIC; 1 nmol) into the same region, to reduce ongoing GABA tone, increased reflex voiding frequency (+467%, n = 16) and tonic activity in the EUS (+56%, n = 7) whilst bursting activity in the EUS became desynchronised. Although muscimol failed to change reflex micturition when microinjected into the dorsal caudal PAG, microinjection of BIC at these sites evoked pronounced autonomic arousal and increased reflex voiding frequency (+237%, n = 34). The results demonstrate that the functional integrity of synapses in the caudal ventrolateral PAG is essential to permit micturition. Transmission through the region is normally regulated by a tonic GABAergic inhibitory influence. In contrast, the functional integrity of the dorsal caudal PAG is not essential for reflex micturition. However, micturition may be initiated from this region via projections to the caudal ventrolateral PAG, as part of the behavioural response to psychological threat or other stressful stimuli.

Progesterone withdrawal-evoked plasticity of neural function in the female periaqueductal grey matter.

Cyclical changes in production of neuroactive steroids during the oestrous cycle induce significant changes in GABA(A) receptor expression in female rats. In the periaqueductal grey (PAG) matter, upregulation of alpha4beta1delta GABA(A) receptors occurs as progesterone levels fall during late dioestrus (LD) or during withdrawal from an exogenous progesterone dosing regime. The new receptors are likely to be extrasynaptically located on the GABAergic interneurone population and to mediate tonic currents. Electrophysiological studies showed that when alpha4beta1delta GABA(A) receptor expression was increased, the excitability of the output neurones in the PAG increased, due to a decrease in the level of ongoing inhibitory tone from the GABAergic interneurones. The functional consequences in terms of nociceptive processing were investigated in conscious rats. Baseline tail flick latencies were similar in all rats. However, acute exposure to mild vibration stress evoked hyperalgesia in rats in LD and after progesterone withdrawal, in line with the upregulation of alpha4beta1delta GABA(A) receptor expression.

Pro-nociceptive action of cholecystokinin in the periaqueductal grey: a role in neuropathic and anxiety-induced hyperalgesic states.

The perception of pain is a dynamic process that is subject to ongoing modulation by central pro- and anti-nociceptive control systems. Diverse factors that may be genetic, gender specific, environmentally determined, psychological or, indeed, engendered by already existing pain-related neuronal activity influence the level of descending control on spinal nociceptive processing. In particular, pain is exacerbated by anxiety. This review examines the evidence for cholecystokinin (CCK)-evoked activation of descending pro-nociceptive facilitatory pathways from the midbrain periaqueductal grey matter (PAG) in mediating anxiety-induced hyperalgesia as well as in the development and maintenance of hyperalgesia associated with peripheral neuropathy. CCK drives a spinal-PAG-medullo-spinal pro-nociceptive positive feedback loop that potentiates spinal transmission of nociceptive afferent input, whilst at the same time suppressing activity in the opioid-driven anti-nociceptive descending pathway from the PAG. In females, responsiveness of PAG neurones to CCK is further modulated by changes in the levels of circulating ovarian hormones, an effect that could underlie the changes in pain sensitivity and responsiveness to opiates that occur during the menstrual cycle and postpartum period.

GABA in the female brain -- oestrous cycle-related changes in GABAergic function in the periaqueductal grey matter.

In many women, aversive psychological and somatic symptoms develop during the late luteal phase of the menstrual cycle, when progesterone levels fall sharply. Following intravenous administration in anaesthetised rats, the progesterone metabolite allopregnanolone readily gained access to the periaqueductal grey (PAG), a region involved in generating panic-like anxiety, and inhibited neural activity via actions at GABA(A) receptors. Withdrawal of female rats from prolonged systemic dosing with progesterone leads to increased numbers of alpha4, beta1 and delta GABA(A) receptor subunit-immunoreactive neurones in the PAG. In naturally cycling rats a similar upregulation occurred during late dioestrus, when progesterone levels fall. Functional experiments revealed that upregulation of alpha4beta1delta receptor subunit expression was associated with a decrease in GABAergic tone in the PAG and increased responsiveness to a panicogenic CCK(2) receptor agonist. The oestrous cycle-linked plasticity of GABA receptors was absent in rats housed in quiet conditions in an isolated room suggesting that environmental factors may be able to influence the central response to hormonal changes. In susceptible animals, i.e. those housed in a communal animal holding room, oestrous cycle-related changes in GABAergic circuits may underlie the development of increased anxiety levels that represent a rodent counterpart to premenstrual syndrome in women.

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.

Cardiovascular and respiratory responses to a panicogenic agent in anaesthetised female Wistar rats at different stages of the oestrous cycle.

In urethane-anaesthetised female Wistar rats, intravenous injection of the panicogenic CCK(B) receptor agonist pentagastrin (0.002-80 microg/kg) evoked a dose-related increase in blood pressure, heart rate and ventilation. The response was blocked in the presence of the selective CCK(B) receptor antagonist CR2945 (1 mg/kg i.v.). The same pattern of cardiovascular and respiratory changes was evoked by microinjection of pentagastrin (0.3 nmol in 250 nL) into the dorsal half of the periaqueductal grey matter (PAG). The effect of intra-PAG administration of pentagastrin was also abolished following injection of CR2945 (1 mg/kg, i.v.). Responsiveness to systemically administered pentagastrin was enhanced in rats in late dioestrus. At the highest dose tested (80 microg/kg), the pressor response, tachycardia and tachypnoea evoked in rats in late dioestrus was significantly higher than rats in proestrus. For rats in oestrus, the pressor response and tachycardia but not tachypnoea were also significantly larger than the response evoked in rats in early dioestrus. The results suggest that the dorsal half of the PAG (dPAG) plays a key role in mediating the cardiovascular and respiratory responses evoked by systemically administered CCK(B) agonists. The enhanced responsiveness to panicogenic agents during late dioestrus may be related to changes in the functional responsiveness of gamma-aminobutyric acid (GABA)ergic circuitry in the dPAG due to plasticity of GABA(A) receptor subunit expression as a consequence of falling progesterone levels.

Plasticity of GABAA receptor subunit expression during the oestrous cycle of the rat: implications for premenstrual syndrome in women.

Many women experience psychological changes during the luteal phase of their menstrual cycle. The late luteal (premenstrual) phase, when symptoms become most severe, is characterized by declining levels of ovarian progesterone. In female rats, withdrawal from prolonged dosing with progesterone leads to upregulation of alpha4 and delta subunits of the GABAA receptor in several brain regions. During the oestrous cycle of the rat, the natural fall in progesterone that occurs in late dioestrus is associated with a parallel increase in expression of alpha4, beta1 and delta GABAA receptor subunits in neurones in the periaqueductal grey matter (PAG), suggesting that new receptors of the alpha4beta1delta composition have been formed. Recombinant alpha4beta1delta receptors display a low EC50 for GABA, which is consistent with activation by extracellular levels of GABA. They are also likely to be located extrasynaptically and to carry tonic currents. In the PAG, a region involved in mediating panic-like anxiety, alpha4, beta1 and delta GABAA receptor subunits are located principally on GABAergic interneurones. On-going GABAergic neuronal activity normally limits and controls the excitability of the panic circuitry. During late dioestrus, when expression of alpha4, beta1 and delta subunits on GABAergic interneurones is upregulated, the increase in tonic current would be expected to lead to a reduction in the activity of the GABAergic population. Thus the panic circuitry would become intrinsically more excitable. It is suggested that during the menstrual cycle in women, plasticity of GABAA receptor subunit expression in brain regions such as the PAG, which are involved in mediating anxiety behaviour, may underlie some of the changes in mood that occur during the premenstrual period.

GABAergic neurones in the rat periaqueductal grey matter express alpha4, beta1 and delta GABAA receptor subunits: plasticity of expression during the estrous cycle.

Immunoreactivity for alpha4, beta1 and delta GABAA receptor subunits on neurones in the periaqueductal gray matter was investigated at different stages of the estrous cycle in Wistar rats. Immunostaining for alpha4, beta1 and delta 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 alpha4, beta1 and delta 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 alpha4, beta1 and delta GABAA receptor subunits almost doubled. We propose that GABAA receptors with the alpha4beta1delta 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.

Changes in GABA(A) receptor subunit expression in the midbrain during the oestrous cycle in Wistar rats.

In women, the late luteal phase or "premenstrual" period is commonly associated with psychological disturbances, which include mood changes and increased aggression. The underlying cause is unknown but one possibility is that fluctuations in levels of neuroactive steroids precipitate changes in expression of GABA(A) receptor subunits that result in functional changes in inhibitory control systems. The present study investigated the levels of expression of alpha4, beta1 and delta GABA(A) receptor subunits in the periaqueductal gray matter (PAG) in rats and whether plasticity occurs during the oestrous cycle in females. In male rats alpha4, beta1 and delta subunit immunoreactive neurones were present throughout the PAG in similar numbers. In female rats in proestrus, oestrus and early dioestrus, the density of alpha4, beta1 and delta subunit immunoreactive cells was similar to males. However, in late dioestrus, the numbers increased significantly, especially in the dorsolateral PAG, a region which is particularly rich in GABAergic interneurones. These parallel changes may reflect an increase in expression of the alpha4beta1delta GABA(A) receptor subtype. Recombinant alpha4beta1delta receptors, expressed in Xenopus oocytes, exhibited and EC(50) for GABA an order of magnitude lower (2.02+/-0.33 microM; mean+/-S.E.M.) than that found for the most ubiquitous alpha1beta2gamma2 GABA(A) receptor (32.8+/-2.5 microM). Increased expression of alpha4beta1delta GABA(A) receptors in the interneurones of the PAG could render the panic circuitry abnormally excitable by disinhibiting the ongoing GABAergic inhibition. Similar changes in neuronal excitability within the PAG in women consequent to falling steroid levels in the late luteal phase of the menstrual cycle could contribute to the development of pre-menstrual dysphoria.

Neuronal activity-related coupling in cortical arterioles: involvement of astrocyte-derived factors.

Neuronal activity-evoked dilatation was investigated in cortical arterioles in brain slices from mature rats maintained in vitro at 31-33 degrees C. In the presence of the thromboxane A2 agonist U46619 (75 nM) to preconstrict vessels, internal diameter decreased by 14.2% and rhythmic contractile activity (vasomotion) developed. Addition of the epoxygenase inhibitor miconazole (20 microm) produced a further decrease in diameter and increase in the frequency of vasomotion, suggesting that tonic release of epoxygenase products maintains a level of cerebrovascular dilator tone. Addition of 1 mum AMPA for 5 min evoked a 15.4 +/- 3.7% increase in diameter and the frequency of vasomotion decreased by -6.7 +/- 1.4 contractions min(-1). The response persisted in the presence of 1 mum TTX, indicating that it was independent of neuronal activity and thus likely to have been evoked by activation of AMPA receptors on astrocytes rather than neurones. The response to the brief (5 min) application of AMPA remained unchanged in the presence of miconazole (20 microm). Prolonged (30 min) application of AMPA produced a +12.1 +/- 1.5% increase in internal diameter and reduction in vasomotion (-8.4 +/- 1.7 contractions min(-1)) that were sustained throughout the stimulation period. However, when AMPA was applied in the presence of miconazole (20 microm) it evoked only a transient increase in diameter (+9.8 +/- 3.1%) and decrease in vasomotion (-6.6 +/- 1.5 contractions min(-1)) that lasted for less than 10 min despite continued application of AMPA. The results suggest that products of epoxygenase activity, probably epoxyeicosatrienoic acids (EETs) are involved in activity-related dilatation in cortical arterioles. Whilst epoxygenase activity is not required to initiate dilatation, it appears to be involved in sustaining the response. Thus EETs released from membrane stores could contribute to the initial stages, but once these have been depleted de novo synthesis of EETs is required to maintain the effect.

Co-localization of 5-HT 2A -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.

Inhibition of vasomotion in hippocampal cerebral arterioles during increases in neuronal activity.

The activity of small arterioles, internal diameter 9.9 +/- 0.8 microm (SEM), was investigated in the CA1 region of hippocampal slices maintained in vitro at 34 degrees C. Under resting conditions, the vessels were quiescent. However, in the presence of the thromboxane A2 agonist U46619 (75-100 nM), rhythmic contractile activity (vasomotion, 1.1-9.9 min(-1), mean 4.1 +/- 0.7 min(-1) SEM) developed in the smooth muscle cells of the vessel walls. Electrical stimulation of the Schaffer collateral fibre pathway was used to evoke increases in neuronal activity in CA1 in the vicinity of the vessels under investigation. A 3-min period of electrical stimulation of the Schaffer collateral fibre pathway produced a significant reduction in vasomotion in 8/8 vessels. During stimulation, vasomotion either ceased completely (n = 5) or the frequency decreased from 7.1, 3.3 and 3.2 min(-1) to 1.2, 0.4 and 0.6 min(-1), respectively (n = 3). In addition, the amplitude of the residual contractions was reduced by 66%, 12% and 52%. In the presence of 1 microM tetrodotoxin (TTX) (n = 4) to block the generation of action potentials, vasomotion was still present. However, the inhibition of vasomotion evoked by increased neuronal activity was blocked concomitant with the abolition of the field potentials recorded in CA1 in response to the stimulation of the Schaffer collaterals. These findings suggest that a reduction in vasomotion may contribute to the local hyperaemia, which accompanies increases in synaptic activity in the brain.

Involvement of GABA in medullary raphe-evoked modulation of neuronal activity in the periaqueductal grey matter in the rat.

Experiments were carried out in urethane-anaesthetised rats to investigate whether GABA is involved in mediating inhibition of neuronal activity in the dorsal half of the periaqueductal grey matter (PAG) after stimulation of the serotonin-containing projection to the PAG from nucleus raphe obscurus (NRO). Multibarrelled micropipettes were used to make recordings from 42 neurones in the dorsal half of the PAG. Most (n = 36) cells were quiescent. Their firing rate was therefore raised to 10-16 Hz by continuous iontophoretic application of DL-homocysteic acid (DLH) in order to facilitate the study of inhibitory events. Iontophoretic application of GABA (0-10 nA) silenced every neurone tested (n = 42), and the effect was blocked by the GABAA antagonist bicuculline (BIC, 10-20 nA; 15/15 cells). BIC also produced an increase in ongoing activity in 14 of 15 cells, indicating the presence of inhibitory GABAergic tone. Iontophoretically applied serotonin (5-HT; 10-70 nA) also inhibited ongoing activity in 9 of 11 cells. The effect of 5-HT was not blocked by BIC. In six of seven cells, microinjection of 100-200 nl DLH into NRO produced a 72.3 +/- 9.4% decrease in neuronal firing rate which was maximal 112 +/- 18 s after the start of the injection and lasted for a total of 313 +/- 63 s. In five of six cells, the raphe-evoked inhibition was blocked by BIC. It is suggested that activation of the serotonergic projection to the PAG from NRO engages GABA-containing interneurones within the PAG which mediate the inhibitory effects of raphe stimulation.