Cortical acetylcholine release and electroencephalogram activation evoked by ionotropic glutamate receptor agonists in the rat basal forebrain

To determine the sensitivity of basal forebrain cholinergic neurons to ionotropic glutamate receptor activation, acetylcholine was collected from the cerebral cortex of urethane-anesthetized rats using microdialysis while monitoring cortical electroencephalographic (EEG) activity. alpha-Amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA; 1, 10, or 100 microM), N-methyl-D-aspartate (NMDA; 100 or 1000 microM) or a combination of AMPA (10 microM) and NMDA (100 microM) was administered to the basal forebrain using reverse microdialysis. Both glutamate receptor agonists produced concentration-dependent, several-fold increases in acetylcholine release indicating that they activated basal forebrain cholinergic neurons; AMPA was more potent, increasing acetylcholine release at a lower concentration than NMDA. The combination of AMPA and NMDA did not produce any greater release than each drug alone, indicating that the effects of these two drugs on cholinergic neurons are not additive. EEG was analyzed by fast Fourier transforms to determine the extent of physiological activation of the cortex. The highest concentrations of AMPA and NMDA tested produced small (25%) but significant increases in high frequency activity. There was a positive correlation across animals between the increases in power in the beta (14-30 Hz) and gamma (30-58 Hz) ranges and increases in acetylcholine release. These results indicate that glutamate can activate cholinergic basal forebrain neurons via both AMPA and NMDA ionotropic receptors but has a more modest effect on EEG activation.     

Tonic activation of NMDA receptors by ambient glutamate of non-synaptic origin in the rat hippocampus

In several neuronal types of the CNS, glutamate and GABA receptors mediate a persistent current which reflects the presence of a low concentration of transmitters in the extracellular space. Here, we further characterize the tonic current mediated by ambient glutamate in rat hippocampal slices. A tonic current of small amplitude (53.99 ± 6.48 pA at +40 mV) with the voltage dependency and the pharmacology of NMDA receptors (NMDARs) was detected in virtually all pyramidal cells of the CA1 and subiculum areas. Manipulations aiming at increasing d -serine or glycine extracellular concentrations failed to modify this current indicating that the glycine binding sites of the NMDARs mediating the tonic current were saturated. In contrast, non-transportable inhibitors of glutamate transporters increased the amplitude of this tonic current, indicating that the extracellular concentration of glutamate primarily regulates its magnitude. Neither AMPA/kainate receptors nor metabotropic glutamate receptors contributed significantly to this tonic excitation of pyramidal neurons. In the presence of glutamate transporter inhibitors, however, a significant proportion of the tonic conductance was mediated by AMPA receptors. The tonic current was unaffected when inhibiting vesicular release of transmitters from neurons but was increased upon inhibition of the enzyme converting glutamate in glutamine in glial cells. These observations indicate that ambient glutamate is mainly of glial origin. Finally, experiments with the use-dependent antagonist MK801 indicated that NMDARs mediating the tonic conductance are probably extra-synaptic NMDARs.     

Extracellular K+ accumulations and synchronous GABA-mediated potentials evoked by 4-aminopyridine in the adult rat hippocampus

Transient changes in extracellular potassium concentration ([K⁺]₀) and field potentials were evoked by 4-aminopyridine (4-AP; 50–100 M) and recorded with ion-selective microelectrodes in CA1b, CA3b and dentate sectors of adult rat hippocampal slices. Long-lasting field potentials recurred at a frequency of 1/60 s (0.016±0.003 Hz) in association with increases in [K⁺]₀ which were largest and most sustained in the dendritic regions where afferent fibers terminate (dentate>CAl>CA3) and in the hilus. In stratum radiatum of CA1 or stratum moleculare of the dentate these fields had a peak amplitude of 1.4±0.29 mV, duration 8.3±1.6 s, and were accompanied by increases in [K⁺]₀ of 1.8±0.22 mM that lasted 32±5.5 s (n = 17 slices). Interictal epileptiform potentials, which were brief (<0.2 s) and more frequent at 1/3 s (0.30±0.02 Hz) were also present in CA1, CA3 and the hilus and associated with small increases in [K⁺]₀ (0.5 mM, duration 2 s). Interictal activity was blocked by 6-cyano-7-nitroquinoxalone-2,3-dione (CNQX; 5–20 M); the slow, less frequent potentials were resistant to both CNQX and dl-2amino-5-phosphonovaleric acid (APV; 50 M) and reversibly blocked (or attenuated by 80%) by bicuculline methiodide (BMI) (25–100 M). The BMI-sensitive potentials were also abolished by baclofen (100 M), an effect which was reversed by 2-OH-saclofen (100 M). Focal application of KCl or GABA in the absence of 4-AP evoked long-lasting field and [K⁺]₀ potentials which were similar to those evoked by 4-AP but more sustained. The proportional relationship between the amplitudes of field and K⁺ potentials with GABA closely resembled that observed for 4-AP; in contrast the slope of KCl-evoked responses was lower. Our results demonstrate that in the adult rat hippocampus 4-AP induces in many different regions accumulations of [K⁺]₀ in synchrony with the long-lasting field potentials, which are known to correspond to an intracellular long-lasting depolarization of the pyramidal cells. These changes are smaller than those which occur in the immature rat hippocampus — which may be related to differences in Na-K-ATPase and susceptibility to seizures. These events involve the activation of GABA_A receptors, are under the modulatory control of GABA_B receptors, and likely arise from the activity of GABAergic interneurons and/or afferent terminals. The long-lasting field potentials appear to reflect mainly the direct depolarizing actions of GABA and to a much more limited extent the associated accumulation of [K⁺]₀.     

Selective modulation of excitatory transmission by mu-opioid receptor activation in rat supraoptic neurons

Opioid peptides have profound inhibitory effects on the production of oxytocin and vasopressin, but their direct effects on magnocellular neuroendocrine neurons appear to be relatively weak. We tested whether a presynaptic mechanism is involved in this inhibition. The effects of mu-opioid receptor agonist D-Ala(2), N-CH(3)-Phe(4), Gly(5)-ol-enkephalin (DAGO) on excitatory and inhibitory transmission were studied in supraoptic nucleus (SON) neurons from rat hypothalamic slices using whole cell recording. DAGO reduced the amplitude of evoked glutamatergic excitatory postsynaptic currents (EPSCs) in a dose-dependent manner. In the presence of tetrodotoxin (TTX) to block spike activity, DAGO also reduced the frequency of spontaneous miniature EPSCs without altering their amplitude distribution, rising time, or decaying time constant. The above effects of DAGO were reversed by wash out, or by addition of opioid receptor antagonist naloxone or selective mu-antagonist Cys(2)-Tyr(3)-Orn(5)-Pen(7)-NH(2) (CTOP). In contrast, DAGO had no significant effect on the evoked and spontaneous miniature GABAergic inhibitory postsynaptic currents (IPSCs) in most SON neurons. A direct membrane hyperpolarization of SON neurons was not detected in the presence of DAGO. These results indicate that mu-opioid receptor activation selectively inhibits excitatory activity in SON neurons via a presynaptic mechanism.     

Reduction of excitatory postsynaptic responses by persistently active metabotropic glutamate receptors in the hippocampus

The release of glutamate from axon terminals is under the control of a variety of presynaptic receptors, including several metabotropic glutamate receptors (mGluRs). Synaptically released glutamate can activate mGluRs within the same synapse where it was released and also at a distance following its diffusion from the synaptic cleft. It is unknown, however, whether the release of glutamate is under the control of persistently active mGluRs. We tested the contribution of mGluR activation to the excitatory postsynaptic responses recorded from several types of GABAergic interneuron in strata oriens/alveus of the mouse hippocampus. The application of 1 microM (alphaS)-alpha-amino-alpha-[(1S,2S)-2-carboxycyclopropyl]xanthine-9-propanoic acid (LY341495), a broad-spectrum mGluR (subtypes 2/3/7/8) antagonist at this concentration, increased evoked-excitatory postsynaptic current (eEPSC) amplitudes by 60% (n = 33). On identified cell types, LY341495 had either no effect (7 of 14 basket and 7 of 13 oriens-lacunosum moleculare, O-LM cells) or resulted in a 32 +/- 30% (mean +/- SD) increase in EPSC amplitudes recorded from basket cells and a seven-times greater (216 +/- 102%) enhancement of EPSCs in O-LM cells. The enhancement of the first EPSC of a high-frequency train indicates persistent mGluR activation. During antagonist application, the relative increase in EPSC amplitude evoked by the second and subsequent pulses in the train was not larger than that of the first EPSC, showing no further receptor activation by the released transmitter. The effect of mGluR subtype selective agonists [3 microM L(+)-2-amino-4-phosphonobutyric acid (L-AP4): mGluR4/8; 600 microM L-AP4: mGluR4/7/8; 1 microM (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IU): mGluR2/3] and an antagonist (0.2 microM LY341495: mGluR2/3/8) suggests that persistently active mGluR2/3/8 control the excitability of hippocampal network.     

Extracellular accumulation of glutamate in the hippocampus induced by ischemia is not calcium dependent--in vitro and in vivo evidence

Calcium dependency of ischemia-induced increase in extracellular glutamate in the hippocampus was studied in vitro and in vivo. Perfusion of a low pO2 medium without glucose (in vitro ischemia) induced an increase in extracellular glutamate in rat hippocampal slices. This increase did not depend on Ca2+, which is in contrast with the observation that about 40% of membrane depolarization (50 mM KCl)-evoked release was Ca2+-dependent. In vivo cerebral ischemia of 5 min duration in gerbils also caused Ca2+-independent increase in extracellular glutamate in the hippocampus. The data suggest that the increase in extracellular glutamate induced by ischemia is not due to the enhanced release of neurotransmitter glutamate.     

Group II metabotropic glutamate receptor modulation of excitatory transmission in rat subthalamic nucleus

Patch pipettes were used to record currents in whole-cell configuration to study the effects of group II metabotropic glutamate receptor (mGluR) stimulation on synaptic transmission in slices of rat subthalamic nucleus. Evoked glutamatergic excitatory postsynaptic currents (EPSCs) were reversibly reduced by the selective group II mGluR agonist (2' S ,2' R ,3' R )-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) in a concentration-dependent manner, with an IC₅₀ of 0.19 ± 0.05 µ m . DCG IV (1 µ m ) had no effect on inhibitory postsynaptic currents mediated by GABA. DCG IV-induced inhibition of EPSCs was reversed by the selective group II mGluR antagonist LY 341495 (100 n m ) and mimicked by another selective group II agonist (2 S ,1' S ,2' S )-2-(carboxycyclopropyl)glycine ( l -CCG-I). Inhibition of EPSC amplitude by DCG IV and l -CCG-I was associated with an increase in the paired-pulse ratio of EPSCs. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (2 µ m ) reduced the inhibitory effect of DCG IV on EPSCs. However, the response to DCG IV was not affected by the protein kinase A (PKA) activator forskolin (20 µ m ), by the adenylyl cyclase inhibitor MDL 12230A (20 µ m ), or by the phosphodiesterase inhibitor Ro 20–1724 (50 µ m ). DCG IV-induced inhibition of EPSCs was reduced by the non-selective protein kinase inhibitors H-7 (100 µ m ), H-8 (50 µ m ) and HA-1004 (100 µ m ). These results suggest that group II mGluR stimulation acts presynaptically to inhibit glutamate release by a PKC-dependent mechanism in the subthalamic nucleus.     

Involvement of glutamate neurotransmission and N-methyl-d-aspartate receptor in the activation of midbrain dopamine neurons by 5-HT1A receptor agonists: an electrophysiological study in the rat

Systemic administration of selective 5-HT1A agonists, such as 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OHDPAT), stimulates the electrical activity of ventral tegmental area (VTA) dopamine neurons by a mechanism which remains unknown. We have examined if this activation is dependent on glutamatergic, serotonergic and GABAergic neurotransmission and if 5-HT1A receptors located within the VTA or within the prefrontal cortex (PFC) could contribute. In vivo electrophysiological recordings were obtained from VTA dopamine neurons from anesthetized rats. The i.v. administration of the 5-HT1A agonist 8-OHDPAT induced a strong stimulation of burst and firing activity of dopamine neurons. This activation remained unchanged in rats pre-treated with the 5-HT depleting agent parachlorophenylalanine. However, pre-administration of the GABAB receptor antagonist phaclophen, but not of the GABAA antagonist picrotoxin, significantly reduced the 8-OHDPAT-induced activation. The N-methyl-d-aspartate (NMDA) antagonist MK 801 (dizocilpine), but not the AMPA/kainate antagonist [1,2,3,4-tetrahydro-7-morpholinyl-2,3-dioxo-6-(fluoromethyl)quinoxalin-1-yl] methyl-phosphonate (ZK 200775), partially prevented or reversed the effects of 8-OHDPAT. However, only the combined pre-administration of the two glutamate antagonists did completely prevent the activatory response to 8-OHDPAT and even converted the effect of 8-OHDPAT into an inhibition, in half of the dopamine neurons tested. Inactivation of the local 5-HT1A receptors by the microinfusion within the VTA of the selective 5-HT1A antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate (WAY 100,635), or of pertussis toxin, reduced the ability of 8-OHDPAT to stimulate the firing of dopamine neurons but not their burst activity. On the other hand, burst activation elicited by 8-OHDPAT was strongly reduced following the inactivation of prefrontal 5-HT1A receptors achieved by the microinfusion of WAY 100,635 within the PFC. These results show that activation of midbrain dopamine neurons by the systemic administration of 5-HT1A agonists does involve the inactivation of a tonic GABAergic tone, involving mainly the GABAB receptors, probably leading to the stimulation of a glutamatergic excitatory drive from the PFC to the VTA and an increase in glutamate release. This will excite dopamine neurons, preferentially through NMDA receptors. Furthermore, our results suggest that some 5-HT1A receptors located within the VTA may also participate in this activation.     

Presynaptic modulation of glutamate and dynorphin release by excitatory amino acids in the guinea-pig hippocampus.

Excitatory amino acid agonists and antagonists were evaluated for their ability to affect the concomitant release of endogenous L-glutamate and dynorphin A(1-8)-like immunoreactivity from guinea-pig hippocampal mossy fiber synaptosomes. Previous work in this laboratory demonstrated that L(+)2-amino-4-phosphonobutyrate inhibits the potassium-evoked release of these endogenous neurotransmitters from guinea-pig but not rat hippocampal mossy fiber synaptosomes. Therefore, the present study was conducted to evaluate excitatory amino acid agonists as indices to the functional properties of this L(+)2-amino-4-phosphonobutyrate-sensitive glutamatergic autoreceptor on mossy fiber terminals. Low micromolar concentrations of quisqualate, but not kainate, N-methyl-D-aspartate, nor RS-alpha-amino-3-hydroxy-5-methyl-4-isoazole-propionic acid, significantly inhibited the potassium-evoked release of both L-glutamate and dynorphin A(1-8)-like immunoreactivity. Quisqualate-induced inhibition of L-glutamate release from mossy fiber terminals was antagonized by the non-N-methyl-D-aspartate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. In contrast, high concentrations of kainate enhanced the potassium-evoked release of L-glutamate and dynorphin A(1-8)-like immunoreactivity, and this potentiation was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. Kainate (1 mM) was the only agonist which significantly enhanced the basal release of L-glutamate, whereas the spontaneous efflux of dynorphin A(1-8)-like immunoreactivity was not affected by any of the agonists tested. The results presented in this paper suggest the existence of inhibitory and excitatory presynaptic glutamatergic autoreceptors that act to modulate the release of endogenous L-glutamate- and prodynorphin-derived peptides from guinea-pig hippocampal mossy fiber terminals. These inhibitory and excitatory autoreceptors, which are sensitive to quisqualate/L(+)2-amino-4-phosphonobutyrate or kainate, respectively, may play an important role in regulating synaptic activity at glutamatergic synapses throughout the central nervous system.     

Group II metabotropic glutamate receptor activation is required for normal hippocampal mossy fibre development in the rat

Glutamate is the main neurotransmitter at hippocampal mossy fibre (MF) terminals. Because neurotransmitters have been proposed as regulating factors of neural network formation and neurite morphogenesis in the developing CNS, we examined the possible contribution of glutamate to MF pathfinding. Entorhino-hippocampal slices prepared from early postnatal rats were cultivated in the presence of glutamate receptor antagonists. Timm histochemical staining revealed that pharmacological blockade of metabotropic glutamate receptors (mGluR), but not of ionotropic glutamate receptors, induced abnormal outgrowth of the MFs. When slices were cultured in the presence of mGluR antagonists, DiI-labelled MF axons displayed a great degree of defasciculation, and MF-mediated EPSPs in the CA3 pyramidal cells were altered. Similar results were obtained for a selective antagonist of group II mGluR, but not of group I or III mGluR. Glutamate is, therefore, likely to regulate MF outgrowth via activation of group II mGluR. The present study may provide a novel role of glutamate in hippocampal development.     

Activation of gonadotropin-releasing hormone receptors induces a long-term enhancement of excitatory postsynaptic currents mediated by ionotropic glutamate receptors in the rat hippocampus

Besides apolipoprotein E (APOE) polymorphism, whose association with Alzheimer's disease (AD) has been confirmed in most of the numerous population samples studied, other markers have been investigated. In most cases the association firstly described was not confirmed in subsequent works. Since it is important to examine these associations in as many populations as possible, we investigated APOE, APOC1, APOC2, alpha-1 antichymotrypsin (ACT) and presenilin-1 (PS-1) polymorphisms in a series of elderly patients with late-onset sporadic AD from Northern Italy and in a sex and age-matched control group. We could not confirm the significantly higher frequency of the ACT*A allele among carriers of APOE e*4 allele described elsewhere, although a similar trend was observed. The APOC2 and the PS-1 distributions were similar between patients and controls. However, we observed a significant difference in the genotype and allele frequencies of APOE and APOC1: patients had higher e*4 and C1*2 allele frequencies. This finding confirms the important role for APOE in AD occurrence. In addition, APOC1 seems to be an interesting marker because, though in strict linkage disequilibrium with APOE, it seems to play an independent role in AD risk. In contrast to previously reported data, plasma apoE concentrations were similar in patients and in controls. An interaction between APOE and APOC1 polymorphisms and apoE levels was observed in patients: subjects carrying the APOE E3/E2 or the APOC1 2-2 genotype have higher apoE concentrations than those who do not.     

Chronic corticosterone administration down-regulates metabotropic glutamate receptor 5 protein expression in the rat hippocampus

Several lines of evidence suggest a dysfunctional glutamate system in major depressive disorder (MDD). Recently, we reported reduced levels of metabotropic glutamate receptor subtype 5 (mGluR5) in postmortem brains in MDD, however the neurobiological mechanisms that induce these abnormalities are unclear. In the present study, we examined the effect of chronic corticosterone (CORT) administration on the expression of mGluR5 protein and mRNA in the rat frontal cortex and hippocampus. Rats were injected with CORT (40 mg/kg s.c.) or vehicled once daily for 21 days. The expression of mGluR5 protein and mRNA was assessed by Western blotting and quantitative real-time PCR (qPCR). In addition, mGluR1 protein was measured in the same animals. The results revealed that while there was a significant reduction (−27%, P=0.0006) in mGluR5 protein expression in the hippocampus from CORT treated rats, mRNA levels were unchanged. Also unchanged were mGluR5 mRNA and protein levels in the frontal cortex and mGluR1 protein levels in both brain regions. Our findings provide the first evidence that chronic CORT exposure regulates the expression of mGluR5 and are in line with previous postmortem and imaging studies showing reduced mGluR5 in MDD. Our findings suggest that elevated levels of glucocorticoids may contribute to impairments in glutamate neurotransmission in MDD.     

GABAB receptor stimulation by baclofen and taurine enhances excitatory amino acid induced phosphatidylinositol turnover in neonatal rat cerebellum.

Excitatory amino acid stimulation of phosphatidylinositol (PI) hydrolysis has been associated with development of the CNS. Normally minimally ineffective in stimulating PI hydrolysis in the neonatal rat cerebellum, N-methyl-D-aspartate (NMDA) increased levels of PI hydrolysis 82.3 +/- 5.5% above basal values in the presence of 1 microM baclofen, a gamma-aminobutyric acidB (GABAB) receptor agonist. This effect was observed at day 7 but not in adult cerebellum. The effect of baclofen could be mimicked by low dose GABA and taurine, actions which were blocked by prior application of a specific GABAB antagonist. Therefore, the ability of NMDA to stimulate PI hydrolysis in neonatal cerebellar tissue may be regulated by the degree of GABAB receptor stimulation.     

Kappa opioid receptor activation depresses excitatory synaptic input to rat locus coeruleus neurons in vitro

Intracellular recordings were made from neurons of the rat locus coeruleus contained within a brain slice maintained in vitro. When applied to the slice in known concentrations the selective kappa opioid receptor agonist trans-(+)-3,4-dichloro-N-methyl-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide methane sulphonate (U50488) (0.01-1 microM) produced a concentration-dependent depression of the excitatory post-synaptic potential evoked by electrical stimulation of afferent inputs to the locus coeruleus. This effect was antagonized by naloxone with an apparent dissociation equilibrium constant (Kd) of 28 nM. U50488 did not completely abolish the EPSP. Over the same concentration range U50488 had no effect on the resting membrane potential, input resistance or action potential waveform of locus coeruleus neurons, nor did U50488 depress the depolarization produced by local application of L-glutamic acid. The mu opioid receptor agonists [D-Ala2, NMe Phe4, Gly-ol5] enkephalin (0.003-1 microM) and [D-Ala2, NMe Phe4, Met(O)5] enkephalinol (0.003-1 microM) caused a membrane hyperpolarization concomitant with a fall in neuronal input resistance. These effects were concentration-dependent and antagonized by naloxone with an apparent Kd of 1.5 nM. Mu agonists also caused a depression of the tetrodotoxin resistant action potential. An in vitro autoradiographic study of [3H]bremazocine binding within the locus coeruleus revealed that, although the majority of binding appears to be to mu sites, a significant proportion was displaceable by unlabelled U50488 and thus represented kappa binding sites. The possibility that kappa opioid receptors may be located pre-synaptically within the locus coeruleus, and that activation of these receptors depresses excitatory synaptic input, is discussed.     

The modulation of excitatory synaptic transmission by adenosine in area CA1 of the rat hippocampus is temperature dependent

We tested the possibility that extracellular adenosine concentration varies with tissue temperature by measuring the tonic adenosinergic inhibition of excitatory synaptic transmission at different temperatures in the in vitro rat hippocampus. Application of the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) enhanced population excitatory postsynaptic potentials (EPSPs) by antagonizing tonic adenosinergic inhibition; this effect was greatest at 25 degrees C, and was progressively reduced at 35 and 37.5 degrees C. These results demonstrate that tonic adenosinergic inhibition is inversely related to temperature. In a second experiment, an exogenous A1 agonist, N6-cyclohexyladenosine (CHA), was applied to slices to inhibit evoked EPSPs. CHA inhibition of EPSPs was greater at 35 than at 25 degrees C, demonstrating that the reduced adenosinergic inhibition at higher temperatures is not a result of reduced A1 receptor function.     

Micturition evoked by glutamate microinjection in the ventrolateral periaqueductal gray is mediated through Barrington's nucleus in the rat

Neural tracing experiments have demonstrated a direct spinal projection to Barrington's nucleus and a possible indirect pathway to Barrington's nucleus via the periaqueductal gray. We sought to identify the role of the periaqueductal gray matter in micturition in urethane-anesthetized rats. Blockade of micturition by focal injection of cobalt chloride was used to identify sites critical to micturition. These sites were located near the ventral margin of the caudal ventrolateral periaqueductal gray and in Barrington's nucleus. L-Glutamate injections into caudal regions of the periaqueductal gray evoked bladder contraction with coordinated sphincter activation. Additional L-glutamate sites with a similar pattern of response and sites where sphincter activation was produced without bladder contraction were found more rostrally and dorsally in the periaqueductal gray. Activation of bladder contractions by L-glutamate injection in the ventrolateral periaqueductal gray was blocked by prior injection of cobalt chloride into Barrington's nucleus.From these data we propose that ventrolateral periaqueductal gray is functionally important to micturition in the urethane-anesthetized rat. Further, we have shown that a periaqueductal gray to Barrington's nucleus pathway is functionally relevant to central mediation of bladder contraction.