Firing modes of midbrain dopamine cells in the freely moving rat

There is a large body of data on the firing properties of dopamine cells in anaesthetised rats or rat brain slices. However, the extent to which these data relate to more natural conditions is uncertain, as there is little quantitative information available on the firing properties of these cells in freely moving rats. We examined this by recording from the midbrain dopamine cell fields using chronically implanted microwire electrodes. (1) In most cases, slowly firing cells with broad action potentials were profoundly inhibited by the dopamine agonist apomorphine, consistent with previously accepted criteria. However, a small group of cells was found that were difficult to classify because of ambiguous combinations of properties. (2) Presumed dopamine cells could be divided into low and high bursting (>40% of their spikes in bursts) groups, with the majority having low bursting rates. The distribution of burst incidence was similar to that previously reported with chloral hydrate anaesthesia, but the average intraburst frequency was higher in the conscious animal at rest and was higher again in bursts triggered by salient stimuli. (3) There was no evidence for spike frequency adaptation within bursts on average, consistent with the hypothesis that afterhyperpolarisation currents may be disabled during behaviourally induced bursting. (4) Presumed dopamine cells responded to reward-related stimuli with increased bursting rates and significantly higher intraburst frequencies compared to bursts emitted outside task context, indicating that modulation of afferent activity might not only trigger bursting, but may also regulate burst intensity. (5) In addition to the irregular single spike and bursting modes we found that extremely regular (clock-like) firing, previously only described for dopamine cells in reduced preparations, can also be expressed in the freely moving animal. (6) Cross-correlation analysis of activity recorded from simultaneously recorded neurones revealed coordinated activity in a quarter of dopamine cell pairs consistent with at least "functional" connectivity. On the other hand, most dopamine cell pairs showed no correlation, leaving open the possibility of functional sub-groupings within the dopamine cell fields.Taken together, the data suggest that the basic firing modes described for dopamine cells in reduced or anaesthetised preparations do reflect natural patterns of activity for these neurones, but also that the details of this activity are dependent upon modulation of afferent inputs by behavioural stimuli.     

Vasodilatation of the hindquarters induced by antagonism of GABA(A) receptors in the freely moving rat

An intracisternal injection of the GABA(A) receptor antagonist bicuculline into conscious rats increased arterial pressure and decreased hindquarter resistance. Propranolol attenuated only the resistance response, but ganglionic block abolished both responses. These findings suggest that central GABA(A) receptor blockade induces an autonomic pressor action with hindquarter vasodilatation.     

Spontaneous REM sleep is modulated by the activation of the pedunculopontine tegmental GABAB receptors in the freely moving rat

Considerable evidence suggests that the neurotransmitter gamma-aminobutyric acid (GABA)-ergic system and pedunculopontine tegmentum (PPT) in the brain stem are critically involved in the regulation of rapid-eye-movement (REM) sleep. GABA and its various receptors are normally present in the PPT cholinergic cell compartment. The aim of this study was to identify the role of GABA and its receptors in the regulation of REM sleep. To achieve this aim, specific receptors were activated differentially by local microinjection of selective GABA receptor agonists into the PPT while quantifying its effects on REM sleep in freely moving chronically instrumented rats (n = 21). The results demonstrated that when GABAB receptors were activated by local microinjection of a GABAB receptor selective agonist, baclofen, spontaneous REM sleep was suppressed in a dose-dependent manner. The optimum dose for REM sleep reduction was 1.5 nmol. In contrast, when GABAA and GABAC receptors were activated by microinjecting their receptor selective agonists, isoguvacine (ISGV) and cis-4-aminocrotonic acid (CACA), respectively, the total percentages of REM sleep did not change compared with the control values. In another eight freely moving rats, effects of baclofen application was tested on firing rates of REM-on cells (n = 12). Of those 12 neurons, 11 stopped firing immediately after application of baclofen [latency: 50 +/- 14 s (SD)] and remained almost silent for 130 +/- 12 min. Findings of the present study provide direct evidence that the PPT GABAB receptors and REM-on cells are involved in the regulation of REM sleep.     

Mu opioid receptors in the ventrolateral periaqueductal gray mediate stress-induced analgesia but not immobility in rat pups

Rat pups become immobile and analgesic when exposed to an adult male rat. The aim of this study was to determine whether these reactions are under the control of endogenous opioids and to determine the role of the midbrain periaqueductal gray (PAG), which mediates stress-induced immobility and analgesia in adult animals. In Experiment 1, 14-day-old rats were injected systemically with the general opioid receptor antagonist naltrexone (1 mg/kg), which blocked male-induced analgesia to thermal stimulation but did not affect immobility. In Experiment 2, the selective mu opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP; 50 or 100 ng/200 nl) was microinjected into the ventrolateral and lateral PAG. CTOP suppressed male-induced analgesia when injected into the ventrolateral PAG. Male-induced immobility was not affected by CTOP. Male proximity therefore seems to induce analgesia in rat pups by releasing endogenous opioids that bind to mu opioid receptors in the ventrolateral PAG.     

Distribution of mu-opioid receptors in rat visceral premotor neurons

Agonists of the mu-opioid receptor (MOR) can modulate the activity of visceral premotor neurons, including cardiac premotor neurons. Neurons in brainstem regions containing these premotor neurons also contain dense concentrations of the MOR1. This study examined the distribution of MOR1 within two populations of visceral premotor neurons: one located in the dorsal motor nucleus of the vagus and the other in the nucleus ambiguus. Visceral premotor neurons contained the retrograde tracer Fluoro-Gold following injections of the tracer into the pericardiac region of the thoracic cavity. MOR1 was localized using immunogold detection of an anti-peptide antibody. Visceral premotor neurons in both regions contained MOR1 at somatic and dendritic sites, although smaller dendrites were less likely to contain the receptor than larger dendrites, suggesting there may be selective trafficking of MOR1 within these neurons. MOR1 labeling in nucleus ambiguus neurons was more likely to be localized to plasma membrane sites, suggesting that ambiguus neurons may be more responsive to opioid ligands than neurons in the dorsal motor nucleus of the vagus. In addition, many of the dendrites of visceral premotor neurons were in direct apposition to other dendrites. MOR1 was often detected at these dendro-dendritic appositions that may be gap junctions. Together these findings indicate that the activity of individual visceral premotor neurons, as well as the coupling between neurons, may be regulated by ligands of the MOR.     

Trace eyeblink conditioning in the freely moving rat: optimizing the conditioning parameters

Trace eyeblink conditioning (EBC) parameters, with an airpuff unconditioned stimulus, were examined in male Fischer 344 X Brown Norway F1 rats. Integrated electromyographic activity from the upper eyelid was recorded. An 8-kHz tone was superior to white noise as a conditioning stimulus. Rats trained with 30 or 50 trials per session showed similar learning. Reversal of environmental lighting had no significant effect. Trace intervals of 0 and 250 ms yielded well-timed conditioned responses (CRs); intervals of 500 ms or more did not. These experiments provide parameters that reliably yield CRs and suggest limits on the temporal processing capabilities of the rat. EBC can thus be used as part of a comprehensive test battery for learning and memory in this species. Physiological recording and pharmacological manipulations may also be done easily. This combination of approaches should facilitate a more complete understanding of learning mechanisms and age-related memory impairments.     

Alteration in hypothalamic monoamine metabolism of freely moving diabetic rat.

Changes in hypothalamic monoamine metabolism were investigated in freely moving streptozotocin (STZ)-induced diabetic rats by using in vivo microdialysis technique. Six weeks later, the animals were implanted with microdialysis probe (molecular weight cut-off index: 12,000-14,000) into the ventromedial portion of the hypothalamus (VMH). The dialysate was collected and loaded onto HPLC to be assayed for norepinephrine (NE), dopamine (DA), serotonin (5-HT) and their metabolites (MHPG, DOPAC and 5-HIAA). The concentration of NE was decreased in the dialysate from the VMH of diabetic rats, whereas there was no significant change in MHPG level. The concentrations of 5-HT and 5-HIAA were reduced in diabetic rats. The DA concentration was obviously increased accompanied by the reduction of DOPAC level. The observed changes in hypothalamic monoamine metabolism, especially the reduced NE release, may play an important role in the induction of hyperphagia in freely moving STZ-induced diabetic rats.     

5-HT3 receptors control dopamine release in the nucleus accumbens of freely moving rats

ICS 205-930, a selective and potent 5-HT3 receptor antagonist applied either systemically, or locally into the ventral tegmental area, antagonized the stimulation of dopamine release in the nucleus accumbens, induced by the subcutaneous administration of morphine. These findings, obtained by the use of brain microdialysis in awake freely-moving rats, demonstrate in vivo a functional role of 5-HT3 receptors in the brain. Since stimulation of dopamine release in the nucleus accumbens is a prerequisite for the expression of the rewarding properties of morphine, its suppression by ICS 205-930 suggests a possible application of 5-HT3 receptor antagonists in the treatment of addiction.     

Coherent spike-wave oscillations in the cortex and subthalamic nucleus of the freely moving rat

The basal ganglia play a critical role in controlling seizures in animal models of idiopathic non-convulsive (absence) epilepsy. Inappropriate output from the substantia nigra pars reticulata (SNr) is known to exacerbate seizures, but the precise neuronal mechanisms underlying abnormal activity in SNr remain unclear. To test the hypothesis that cortical spike-wave oscillations, often considered indicative of absence seizures, propagate to the subthalamic nucleus, an important afferent of SNr, we simultaneously recorded local field potentials from the frontal cortex and subthalamic nucleus of freely moving rats. Spontaneous spike-wave oscillations in cortex (mean dominant frequency of 7.4 Hz) were associated with similar oscillations in the subthalamic nucleus (mean of 7.9 Hz). The power of oscillations at 5-9 Hz was significantly higher during spike-wave activity as compared with rest periods without this activity. Importantly, spike-wave oscillations in cortex and subthalamic nucleus were significantly coherent across a range of frequencies (3-40 Hz), and the dominant (7-8 Hz) oscillatory activity in the subthalamic nucleus typically followed that in cortex with a small time lag (mean of 2.7 ms). In conclusion, these data suggest that ensembles of subthalamic nucleus neurons are rapidly recruited into oscillations during cortical spike-wave activity, thus adding further weight to the importance of the subthalamic nucleus in absence epilepsy. An increase in synchronous oscillatory input from the subthalamic nucleus could thus partly underlie the expression of pathological activity in SNr that could, in turn, aggravate seizures. Finally, these findings also reiterate the importance of oscillations in these circuits in normal behaviour.     

Depletion of central beta-endorphin blocks midbrain stimulation produced analgesia in the freely-moving rat

The present study examines the role of central beta-endorphin in the generation of stimulation-induced analgesia from the ventral midbrain periaqueductal gray of freely-moving rats. Electrical stimulation of the ventral midbrain periaqueductal gray led to an antinociception against noxious heat which gradually subsided post-stimulation over a period of about 15 min. Locomotor effects (ipsilateral rotation) were also seen which were not correlated in intensity with the analgesia and which disappeared immediately with termination of stimulation. There was no indication of any aversive effects. Application of the opioid antagonist, naloxone, 10 min pre-stimulation, strongly attenuated the antinociception without changing basal thresholds. It did not influence the locomotor changes. Bilateral, radiofrequency lesions of the mediobasal arcuate hypothalamus greatly depleted immunoreactive beta-endorphin from brain tissues without affecting its levels in plasma. Lesioned rats showed a pronounced reduction of stimulation-produced antinociception in the absence of any change in basal thresholds; the locomotor effects of stimulation were not influenced. The degree of depletion of immunoreactive-beta-endorphin significantly correlated with the degree of attenuation of antinociception. These data suggest: stimulation of the ventral midbrain periaqueductal gray leads both to an antinociception and locomotor effects in freely-moving rats: these can be clearly dissociated from each other; the antinociception (but not locomotor effects) are mediated by an endogenous opioid sensitive to blockade by naloxone; and central beta-endorphin may be the endogenous opioid mediating stimulation-produced antinociception from the ventral midbrain periaqueductal gray in the rat.     

GABAergic neurons express mu-opioid receptors in the ventrolateral orbital cortex of the rat

Behavioral studies have indicated that GABAergic modulation is involved in the opioid-induced antinociception in the ventrolateral orbital cortex (VLO). The aim of the current study was to examine whether the GABAergic neurons in the rat VLO expressed mu-opioid receptor subtype 1 (MOR1). This study employed immunofluorescence histochemical double-staining technique and showed that a considerable amount of GABA- and MOR1-like immunoreactive neurons existed in layers II-VI in the VLO. Of these GABA-like immunoreactive neurons, 92.0% of them showed MOR1-like immunoreactivities. Similarly, 80.2% of MOR1-like immuoreactive neurons also exhibited GABA-like immunoreactivities. These results provide morphological evidence that opioid-induced antinociception in the VLO might be due to an inhibitory effect by opioid via MOR1 on GABAergic neurons, resulting in disinhibition of VLO projection neurons and leading to activation of the VLO-PAG brainstem descending pain control system to depress the nociceptive inputs at the spinal cord level.     

Cannabinoids mediate analgesia largely via peripheral type 1 cannabinoid receptors in nociceptors

Although endocannabinoids constitute one of the first lines of defense against pain, the anatomical locus and the precise receptor mechanisms underlying cannabinergic modulation of pain are uncertain. Clinical exploitation of the system is severely hindered by the cognitive deficits, memory impairment, motor disturbances and psychotropic effects resulting from the central actions of cannabinoids. We deleted the type 1 cannabinoid receptor (CB1) specifically in nociceptive neurons localized in the peripheral nervous system of mice, preserving its expression in the CNS, and analyzed these genetically modified mice in preclinical models of inflammatory and neuropathic pain. The nociceptor-specific loss of CB1 substantially reduced the analgesia produced by local and systemic, but not intrathecal, delivery of cannabinoids. We conclude that the contribution of CB1-type receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia is paramount, which should enable the development of peripherally acting CB1 analgesic agonists without any central side effects.     

Evidence for the presence of PS-OFF neurons in the ventromedial medulla oblongata of freely moving cats

Summary Recordings were made from 24 PS-OFF neurons, characterized by a slow rhythmic discharge rate during waking and slow wave sleep and a marked decrease in the firing rate during paradoxical sleep, in the ventromedial medulla oblongata of freely moving cats. These neurons were located in either the nuclei raphe magnus and pallidus or the neighboring reticular formation where serotonincontaining neurons are found in the cat. Two types of medullary PS-OFF neurons are described, and the descending projection and slow conduction velocity of some of these neurons are demonstrated.Supported by INSERM (U 52), CNRS (LA 162), and DGRST (Contract no. 79.7.1077)     

Ultrastructural localization of enkephalin and mu-opioid receptors in the rat ventral tegmental area

Enkephalins are endogenous ligands for opioid receptors whose activation potently modulates the output of mesocorticolimbic dopaminergic neurons within the ventral tegmental area. Many of the reinforcing effects of enkephalins in the mesocorticolimbic system are mediated by mu-opioid receptors. To determine the sites for Leu(5)-enkephalin activation of mu-opioid receptors in the ventral tegmental area, we examined the dual electron microscopic immunocytochemical localization of their respective antigens in this region of rat brain. Enkephalin immunoperoxidase reaction product and mu-opioid receptor immunogold-silver labeling showed similar cellular and subcellular distribution in both the paranigral and parabrachial subdivisions of the ventral tegmental area. Enkephalin immunoreactivity was mainly localized in small unmyelinated axons (50.4%) and in axon terminals (40.4%). The majority of these terminals formed symmetric, inhibitory-type synapses, many of which were on dendrites expressing plasmalemmal mu-opioid receptors. Appositional contacts were also often seen between axons or terminals that were differentially labeled for the two antigens. In addition, some of the enkephalin-labeled terminals and a few somatodendritic profiles showed a plasmalemmal or vesicular localization of mu-opioid receptors.Our results indicate that dendritic targets of inhibitory terminals, as well as nearby axon terminals, are potential sites for enkephalin activation of mu-opioid receptors throughout the ventral tegmental area. Moreover, co-localization of enkephalin and mu-opioid receptors in selective neuronal profiles may indicate an autoregulatory role for these receptors or their internalization along with the bound ligand in this brain region.     

Internalization of mu-opioid receptors produced by etorphine in the rat locus coeruleus.

Chronic administration of mu-opioid receptor agonists is known to produce adaptive changes within noradrenergic neurons of the locus coeruleus. Although mu-opioid receptors are densely expressed by locus coeruleus neurons, the effects of acute and chronic administration of agonists on the subcellular distribution of mu-opioid receptors remain poorly understood. Therefore, we examined the ultrastructural distribution of mu-opioid receptor immunoreactivity in the locus coeruleus of rats subjected to either acute morphine, or etorphine, or chronic morphine treatment. In the locus coeruleus of control rats receiving acute saline injections or placebo pellet implants, immunogold-silver labeling for mu-opioid receptors was localized to parasynaptic and extrasynaptic portions of the plasma membranes of perikarya and dendrites. Only 8% of the gold-silver particles analyzed were distributed within the cytoplasm of dendrites and perikarya in vehicle-treated rats. Immunolabeling for mu-opioid receptors was distributed along portions of the plasma membrane that were often apposed by astroglial sheaths. After acute injections of etorphine, there was a dramatic internalization of mu-opioid receptors to intracellular compartments. Quantitative analysis of gold-silver particles indicative of mu-opioid receptors showed that a substantial number of gold particles shifted from the plasma membrane to early endosomes in dendrites from etorphine-treated rats. In dendrites sampled from etorphine-treated rats, 85% of the gold-silver grains indicative of mu-opioid receptor labeling were located in intracellular compartments as compared to 15% that were distributed along the plasma membrane. In animals that received either acute morphine injections or chronic morphine via pellet implantation, no change in the subcellular distribution of immunogold particles indicative of mu-opioid receptors was detected when compared to matched control animals.These results provide the first ultrastructural evidence that mu-opioid receptors are internalized by agonists such as etorphine, but not the partial agonist morphine, in the locus coeruleus.     

The effects of age on eyeblink conditioning in the freely moving Fischer-344 rat

The effects of age were assessed on the ability of male Fischer-344 rats to acquire a classically conditioned eyeblink using the "delay" paradigm. Using a 350 ms white noise conditioning stimulus and a 100 ms coterminating periorbital shock (2 mA, 60 Hz, AC) we have demonstrated that these rats exhibit deficits as early as middle age. Middle-aged and senescent rats (18 and 30 months) exhibited significantly fewer conditioned responses than young rats (3 and 12 months). Since all of the rats responded to a test noise, and there were no differences in threshold to evoke a blink, this result is likely to be due to an associative deficit. Thus, our results indicate that eyeblink conditioning in the freely moving rat is a useful model system for behavioral and neurobiological analyses of the effects of age on associative learning and memory.     

N-methyl-D-aspartate receptor-independent long-term depression and depotentiation in the sensorimotor cortex of the freely moving rat

Bidirectional modifications in synaptic efficacy are central components in recent models of cortical learning and memory, and we previously demonstrated both long-term synaptic potentiation (LTP) and long-term synaptic depression (LTD) in the neocortex of the unanaesthetized adult rat. Here, we have examined the effects of N-methyl-D-aspartate receptor (NMDAR) blockade on the induction of LTD, LTP, and depotentiation of field potentials evoked in sensorimotor cortex by stimulation of the white matter in the adult, freely moving rat. High frequency (300 Hz) stimulation (HFS) was used to induce LTP and prolonged, low-frequency (1 Hz) stimulation was used to induce either depotentiation or LTD. LTD was expressed as a reduction in the amplitude of the short and long-latency field potential components, while depotentiation was expressed as a decrease in the amplitude of a previously enhanced late component. Under NMDAR blockade, HFS failed to induce LTP and instead produced a depression effect similar to LTD. Following washout of the drug, HFS induced a normal LTP effect. Unlike LTP, LTD and depotentiation were found to be NMDAR-independent in the neocortex of the freely moving rat.