Lateral hypothalamus: early developmental expression and response to hypocretin (orexin)

Hypocretin is a recently discovered peptide that is synthesized by neurons in the lateral hypothalamic area (LH) and is believed to play a role in sleep regulation, arousal, endocrine control, and food intake. These functions are critical for the development of independent survival. We investigated the developmental profile of the hypocretin system in rats. Northern blot analysis showed that the expression of hypocretin mRNA increased from postnatal day 1 to adulthood. Both of the identified hypocretin receptor mRNAs were strongly expressed very early in hypothalamic development, and expression subsequently decreased in the mature brain. Immunocytochemistry revealed hypocretin-2 peptide expression in the cell bodies of LH neurons and in axons in the brain and spinal cord as early as embryonic day 19. Whole-cell patch clamp recordings from postnatal P1-P14 LH slices demonstrated a robust increase in synaptic activity in all LH neurons tested (n = 20) with a 383% increase in the frequency of spontaneous activity upon hypocretin-2 (1.5 microM) application. A similar increase in activity was found with hypocretin-1 application to LH slices. Hypocretin-2 evoked a robust increase in synaptic activity even on the earliest day tested, the day of birth. Furthermore, voltage-clamp recordings and calcium digital imaging experiments using cultured LH cells revealed that both hypocretin-1 and -2 induced enhancement of neuronal activity occurred as early as synaptic activity was detected. Thus, as in the adult central nervous system, hypocretin exerts a profound excitatory influence on neuronal activity early in development, which might contribute to the development of arousal, sleep regulation, feeding, and endocrine control.     

Synaptic responses of neurons of the nucleus tractus solitarius in vitro

Postsynaptic responses of neurons in the nucleus tractus solitarius (NTS) have been studied in an in vitro slice preparation using extra- and intracellular recording. Single or paired pulse stimulations were delivered to afferent fibers within the tractus solitarius (TS) to activate orthodromic responses in these neurons. Most NTS neurons displayed an initial synaptic excitation followed by inhibition of spontaneous or evoked firing lasting up to 150-200 ms after stimulation. Excitatory postsynaptic potentials (EPSPs), recorded intracellularly, were increased in amplitude by membrane hyperpolarization. Large afterhyperpolarizations followed action potentials triggered by the EPSPs or evoked by intracellular current injections. Intracellular evidence for synaptic inhibition within the NTS included: (1) the presence, after Cl-injection, of flurries of spontaneous PSPs likely to be inverted inhibitory postsynaptic potentials; (2) reduction of the size of a test EPSP by a previous subthreshold TS conditioning volley; and (3) hyperpolarizing PSPs recorded in some neurons. Other NTS neurons exhibited prolonged excitatory responses to TS stimulation and could be local inhibitory interneurons. These results may help specify synaptic mechanisms in the NTS that could play an integrative role in the relay of visceral sensory inputs to higher order effectors.     

Hypocretin-1 causes G protein activation and increases ACh release in rat pons

The effects of the arousal-promoting peptide hypocretin on brain stem G protein activation and ACh release were examined using 16 adult Sprague-Dawley rats. In vitro[35S]GTPgammaS autoradiography was used to test the hypothesis that hypocretin-1-stimulated G protein activation is concentration-dependent and blocked by the hypocretin receptor antagonist SB-334867. Activated G proteins were quantified in dorsal raphe nucleus (DR), locus coeruleus (LC) and pontine reticular nucleus oral part (PnO) and caudal part (PnC). Concentration-response data revealed a significant (P < 0.001) effect of hypocretin-1 (2-2000 nm) in all brain regions examined. Maximal increases over control levels of [35S]GTPgammaS binding were 37% (DR), 58% (LC), 52% (PnO) and 44% (PnC). SB-334867 (2 micro m) significantly (P < 0.002) blocked hypocretin-1 (200 nm)-stimulated [35S]GTPgammaS binding in all four nuclei. This is the first autoradiographic demonstration that hypocretin-1 activates G proteins in arousal-related brain stem nuclei as a result of specific receptor interactions. This finding suggests that some hypocretin receptors in brain stem couple to inhibitory G proteins. In vivo microdialysis was used to test the hypothesis that PnO administration of hypocretin-1 increases ACh release in PnO. Dialysis delivery of hypocretin-1 (100 micro m) significantly (P < 0.002) increased (87%) ACh release. This finding is consistent with the interpretation that one mechanism by which hypocretin promotes arousal is by enhancing cholinergic neurotransmission in the pontine reticular formation.     

Hypocretins (orexins): clinical impact of the discovery of a neurotransmitter

Hypothalamic excitatory hypocretin (orexin) neurons have been discovered in 1998 and found to have widespread projections to basal forebrain, monoaminergic and cholinergic brainstem, and spinal cord regions. The hypocretin system is influenced both neuronally (e.g. suprachiasmatic nucleus, GABAergic, cholinergic and aminergic brainstem nuclei) as well as metabolically (e.g. glucose, ghrelin, and leptin). Physiologically the hypocretin system has been implicated in the regulation of behaviours that are associated with wakefulness, locomotion, and feeding. A role in REM sleep, neuroendocrine, autonomic and metabolic functions has also been suggested. Pathophysiologically a deficient hypocretin neurotransmission has been found in human narcolepsy and (engineered) animal models of the disorder. Different mechanisms are involved including (1) degeneration of hypocretin neurons (mice), (2) hypocretin ligand deficiency (humans, mice, dogs), (3) hypocretin receptor deficiency (mice, dogs). Reports of low hypocretin-1 cerebrospinal fluid levels in neurologic conditions (e.g. Guillain-Barré syndrome, traumatic brain injury, hypothalamic lesions) with and without sleep-wake disturbances and, on the other hand, observations of normal levels in about 11% of narcoleptics raise questions about the exact nature and pathophysiological base of the link between hypocretin deficiency and clinical manifestations in human narcolepsy.     

Activation of GABAergic pathway by hypocretin in the median raphe nucleus (MRN) mediates stress-induced theta rhythm in rats

The frequency of electroencephalograms (EEGs) is predominant in theta rhythm during stress (e.g., footshock) in rats. Median raphe nucleus (MRN) desynchronizes hippocampal theta waves via activation of GABAergic neurons in the medial septum-diagonal band of Broca (MS-DBB), a theta rhythm pacemaker. Increased hypocretin mediates stress responses in addition to the maintenance of wakefulness. Hypocretin receptors are abundant in the MRN, suggesting a possible role of hypocretin in modulating stress-induced theta rhythm. Our results indicated that the intensity of theta waves was enhanced by footshock and that a hypocretin receptor antagonist (TCS1102) suppressed the footshock-induced theta waves. Administration of hypocretin-1 (1 and 10 μg) and hypocretin-2 (10 μg) directly into the MRN simulated the effect of footshock and significantly increased theta waves. Co-administration of GABA(A) receptor antagonist, bicuculline, into the MRN blocked the increase of theta waves induced by hypocretins or footshock. These results suggested that stress enhances the release of hypocretins, activates GABAergic neurons in the MRN, blocks the ability of MRN to desynchronize theta waves, and subsequently increases the intensity of theta rhythm.     

Intracellular study of rat entopeduncular nucleus neurons in an in vitro slice preparation: response to subthalamic stimulation

Responses of rat entopeduncular nucleus (EP) neurons after stimulation of the subthalamic nucleus (STh) and the morphology of the EP neurons were studied using brain slice preparations. EP neurons were classified into two types based on their electrophysiological properties as reported previously. Of 87 EP neurons, 72 were Type I and the rest were Type II. Synaptic responses to STh stimulation were different in these two cell types. STh stimulation evoked excitatory postsynaptic potentials (EPSPs) followed by strong inhibitory postsynaptic potentials (IPSPs) in Type I neurons and EPSPs without strong IPSPs in Type II neurons. The EPSPs were considered to be monosynaptic because no large change in the latency (1.7 ± 0.5ms) resulted by alteration of stimulus intensity. The EPSPs were reversibly suppressed by kynurenic acid in a dose-dependent manner. Bath application of (+)-tubocurarine (10–50 μM) had no effect on EPSPs or IPSPs. Bath application of bicuculline methiodide (50–100 μM) markedly suppressed IPSPs evoked by STh stimulation and at the same time increased the amplitude and duration of EPSPs without affecting the latency. In the presence of bicuculline methiodide, EPSPs could induce plateau potentials and slow action potentials. Some Type I and Type II neurons were intracellularly labeled by biocytin. Type I neurons were located throughout the EP but Type II neurons were located mainly in the dorsal portion of the EP. Medium sized somata of both Type I and Type II neurons were spine-free and fusiform or round in shape. They had 3–4 thick primary dendrites with diameters of 2–5 μm that branched into thin secondary dendrites. The secondary and tertiary dendrites of Type I neurons were sparsely covered with spines. Dendritic terminals of some Type I neurons had complex arborizations with abundant spines and appendages. The dendrites of Type II neurons were generally smooth and had no complex arborizations at their terminals.     

Electrophysiological alterations in subthalamic neurons after unilateral dopamine depletion in the rat

The present study examined the effects of unilateral 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra pars compacta (SNc) on electrophysiological properties of subthalamic neurons (STN) in adult rats. Most neurons displayed regular spontaneous tonic firing patterns in both control and lesioned animals; however, the percentage of neurons with spontaneous burst firing at hyperpolarized membrane potentials was increased significantly in lesioned animals compared with controls (45% vs. 14% respectively). In the presence of bicuculline, a gamma-aminobutyric acid type A (GABAA) receptor antagonist, electrical stimulation of the internal capsule produced monosynaptic excitatory postsynaptic potentials (EPSPs) in almost all recorded neurons. DA (50 microM) increased the amplitude and/or duration of the EPSPs in neurons from both groups, whereas the DA D1 receptor agonist SKF 81297 (10 microM) produced a significant increase in amplitude and/or duration of EPSPs in neurons from the lesioned group only. This latter increase was blocked by pretreatment with the DA D1 antagonist SCH 23390 (10 microM). These data suggest that unilateral degeneration of DA neurons in the SNc changes firing properties and enhances electrophysiological responsiveness of STN neurons to activation of DA D1 receptors.     

Afferent projections in the spinal accessory nerve to the facial motoneurons of the cat

Stimulation of the accessory nerve evoked polysynaptic excitatory postsynaptic potentials (EPSPs) in the facial nucleus (FN) neurons of anesthetized cats. From the experiments with severance of C1-C3 dorsal roots, it is suggested that accessory afferents enter the brainstem through the accessory nerve. It was also found that stimulation of the solitary tract nucleus produced exclusively monosynaptic EPSPs in the FN neurons and the afferent volleys are most likely to be relayed at the solitary tract nucleus.     

Neural correlates of arousal-induced circadian clock resetting: hypocretin/orexin and the intergeniculate leaflet

In Syrian hamsters, some procedures for stimulating behavioural arousal (e.g. running in a novel wheel and sleep deprivation by gentle handling with minimal activity) markedly phase-advance circadian rhythms when applied during the middle of the daily rest period, while other arousal procedures do not (e.g. physical restraint, caffeine and modafinil). The neural basis for this differential effect of arousal procedures on clock resetting is unknown. We used c-fos expression as a marker for neuronal activation to determine whether these arousal procedures differentially activate two nonphotic inputs to the circadian system, the thalamic intergeniculate leaflet (IGL; a proposed nonphotic gateway to the circadian clock) and the hypothalamic hypocretin system (which depolarizes arousal-related cell groups throughout the brain and innervates both the IGL and the peri-suprachiasmatic nucleus region). c-FOS in hypocretin-1-immunoreactive neurons, in hypothalamic nonhypocretin neurons and in the IGL was significantly increased by novel wheel running, gentle handling and physical restraint, but only weakly by systemic injections of modafinil (300 mg/kg) or caffeine (75 mg/kg), at doses that are strongly alerting. Spatial analysis revealed few regional differences in the percentage of cells double-labelled for hypocretin-1 and c-FOS following each treatment. These results suggest that activation of hypocretin neurons (as in the restraint condition) is not sufficient to induce phase shifts, and that gating of arousal effects on circadian clock phase may be downstream from the hypocretin system and from IGL neurons activated by these procedures.     

Diurnal variation of cerebrospinal fluid hypocretin-1 (Orexin-A) levels in control and depressed subjects.

BACKGROUND: Hypocretins, excitatory neuropeptides at monoaminergic synapses, appear to regulate human sleep-wake cycles. Undetectable cerebrospinal fluid hypocretin-1 levels are seen in narcolepsy, which is frequently associated with secondary depression. Shortened rapid eye movement latency is observed in both narcolepsy and depression. Cerebrospinal fluid hypocretin-1 levels have not been reported in mood disorders. METHODS: We examined hypocretin-1 levels in 14 control and 15 depressed subjects. Cerebrospinal fluid was drawn continuously in supine subjects for 24 hours with an indwelling intrathecal catheter under entrained light-dark conditions. Depressed subjects were studied before and after 5 weeks of sertraline (n=10, three nonresponders) or bupropion (n=5, two nonresponders). RESULTS: Hypocretin-1 levels varied slightly (amplitude 10%) but significantly across the diurnal cycle in control subjects, with amplitude significantly reduced in depression (3%). Levels were lowest at midday, surprising for a hypothetically wake-promoting peptide. Mean hypocretin levels trended higher in depressive than in control subjects. Hypocretin-1 levels decreased modestly but significantly after sertraline (-14%) but not bupropion. CONCLUSIONS: Our results are consistent with previous physiologic findings in depression indicating dampened diurnal variations in hypocretin-1. The finding that sertraline but not bupropion slightly decreased cerebrospinal fluid hypocretin-1 indicates a serotoninergic influence on hypocretin tone.     

Excitatory projection from the interpeduncular nucleas to central superior raphe neurons

The projection from the interpeduncular nucleus (IP) to the central superior raphe nucleus (CS) was studied using intracellular electrophysiologic methods. IP stimulation generates monosynaptic EPSPs in a large number of CS neurons studied with latency of 1–2 ms. Intracellular peroxidase injections into CS neurons responding to IP shock confirmed the location and somatic origin of intracellular potentials. These findings document the existence of a direct excitatory projection from IP onto CS neurons.     

Effects on sleep and wakefulness of the injection of hypocretin-1 (orexin-A) into the laterodorsal tegmental nucleus of the cat

Anatomical data demonstrate a dense projection, in the cat, from hypocretin (orexin) neurons in the hypothalamus to the laterodorsal tegmental nucleus (LDT), which is a critical pontine site that is involved in the regulation of the behavioral states of sleep and wakefulness. The present study was therefore undertaken to explore the hypocretinergic control of neurons in the LDT vis-à-vis these behavioral states. Accordingly, hypocretin-1 was microinjected into the LDT of chronic, unanesthetized cats and its effects on the percentage, latency, frequency and duration of wakefulness, quiet (non-REM) sleep and active (REM) sleep were determined. There was a significant increase in the time spent in wakefulness following the microinjection of hypocretin-1 into the LDT and a significant decrease in the time spent in active sleep. The increase in the percentage of wakefulness was due to an increase in the duration of episodes of wakefulness; the reduction in active sleep was due to a decrease in the frequency of active sleep episodes, but not in their duration. These data indicate that hypocretinergic processes in the LDT play an important role in both of the promotion of wakefulness and the suppression of active sleep.     

Estrogen priming affects active membrane properties of medial amygdala neurons

The medial nucleus of the amygdala (MNA) in the rat is a target tissue for estrogen binding and a sexually dimorphic structure. We used an in vitro slice preparation and intracellular recording techniques to study the effects of β-estradiol priming on active and passive membrane properties of MNA neurons. Two groups of adult female rats were used; ovariectomized (OVX) non-primed rats and OVX rats that were estrogen-primed at least 24 h prior to recording. Estrogen priming increased the occurrence of spontaneous excitatory postsynaptic potentials (EPSPs) in MNA neurons, and of depolarizing afterpotentials (DAPs) observed with a cathodally triggered action potential, and was associated with a lack of accommodation in these cells. Overall, long-term exposure to estrogen markedly increased the spontaneous activity and excitability of the MNA neurons.     

Tectal afferents monosynaptically activate neurons in the pigeon isthmo-optic nucleus

Postsynaptic responses of 105 neurons in brain slices were intracellularly recorded from the isthmo-optic nucleus (ION) in pigeons, and 18 of these neurons were labeled with Lucifer yellow. Excitatory postsynaptic potentials (EPSPs) or spikes were produced in 93 cells, inhibitory postsynaptic potentials (IPSPs) in 10 cells, and EPSPs followed by IPSPs in two cells following electrical stimulation of the tecto-isthmooptic tract. The EPSPs occurred in an all-or-none fashion, with short latencies (1.3 +/- 0.6 ms). Repetitive stimulation increased their amplitude and duration, demonstrating that temporal summation was involved. Neurons producing excitatory responses were distributed throughout cellular layers of the nucleus. Pure IPSPs had a latency of 3.9 +/- 2.3 ms, and cells that responded in this manner were only distributed in the rostral portion of the nucleus. In the remaining two cells with EPSP-IPSP responses, the latency of excitatory responses was 1.5 ms in one cell and 1.4 ms in the other, and that of inhibitory responses was, respectively, 5.1 and 4.1 ms. Thus, it appeared that excitation was monosynaptic, whereas inhibition may be polysynaptic. Four single injections resulted in dye-coupled labeling, and two pairs of closely apposed cells fired spikes, probably resulting from spatial summation of their excitatory responses. The present study suggests that tectal cells directly activate ION neurons and that tectal fibers contact isthmo-optic neurons in a one-to-one fashion. Taken together with previous studies, it appears that the entire tecto-ION-retinal pathway is excitatory.     

Cocaine actions on rat prefrontal cortical and hippocampal dentate granule neurons in vitro

The electrophysiological actions of cocaine hydrochloride (COC) on medial prefrontal cortical (mpfc) and hippocampal dentate granule (DG) neurons were investigated in rat brain slices with intracellular recording techniques. The following parameters were measured: resting membrane potential (RMP), spike threshold, spike firing adaptation, postspike train afterhyperpolarization (AHP), excitatory postsynaptic potentials (EPSPs), and inhibitory postsynaptic potentials (IPSPs). In the mpfc, COC appeared to have both inhibitory and excitatory effects. In the majority of cells examined, the EPSP amplitude was attenuated by COC (200 nM–20 μM), whereas the amplitude of the postspike train afterhyperpolarization (AHP) was reduced (an excitatory effect). In DG neurons, 1 μM COC caused a small depolarization. COC potentiated the EPSPs at 1 μM but attenuated EPSPs and IPSPs at 10–100 μM. The amplitude of antidromically evoked EPSPs was also increased by 20 μM COC. At concentrations of 10 μM and greater, COC increased spike threshold. It is concluded that COC actions on mpfc and DG neurons are both excitatory and inhibitory and that these effects may be mediated by multiple neurotransmitters/modulators. © 1993 Wiley-Liss, Inc.     

Postsynaptic excitation of prefrontal cortical pyramidal neurons by hypocretin-1/orexin A through the inhibition of potassium currents

Hypocretins are crucial for the regulation of wakefulness by the excitatory actions on multiple subcortical arousal systems. To date, there is little information about the direct postsynaptic excitatory effects of hypocretins on the neurons in prefrontal cortex (PFC), which is important for higher cognitive functions and is correlated with level of wakefulness. In this study, we tested the excitatory effects of hypocretin-1 on acutely isolated PFC pyramidal neurons of rats and studied the possible ionic mechanisms by using whole-cell patch-clamp techniques. Puff application of hypocretin-1 caused a dose-dependent excitation. Further observations that perfusion of Ca2+-free artificial cerebrospinal fluid did not influence the depolarizing effects of hypocretin-1, in conjunction with the findings that hypocretin-1 could decrease net whole-cell K+ currents, demonstrate that the excitatory effects of hypocretin-1 on PFC neurons are mediated by the inhibition of K+ currents but not Ca2+ influx. Finally, the decrease in K+ currents induced by hypocretin-1 was abolished by a protein kinase C (PKC) inhibitor (BIS II) or a phospholipase C (PLC) inhibitor (D609), suggesting that PKC and PLC appear to be involved in mediating the inhibitory effects of hypocretin-1 on K+ currents. These results indicate that hypocretin-1 exerts a postsynaptic excitatory action on PFC neurons through the inhibition of K+ currents, which probably results from activation of PKC and PLC signaling pathways.     

Spinal dorsal horn neurons in elevated extracellular calcium: cell properties and spontaneous discharges

Recordings were made from neurons in the dorsal horn (DH), and from dorsal and ventral roots (DRs and VRs) of isolated spinal cords of infant mice. Raising calcium concentration ([Ca2+]) in the organ bath from 1.2 to 2.4 mmol/l resulted in a slight hyperpolarization, elevation of threshold current (rheobase), and augmentation of excitatory postsynaptic potentials (EPSPs). In many cells EPSPs acquired a much prolonged late phase. Orthodromic stimulation evoked in some DH neurons an action potential that had the same threshold as, and coincided in time with, the 'dorsal horn response' (DHR) recorded from DR. In spinal cords bathed in elevated [Ca2+], DR recordings showed irregularly recurring spontaneous waves, and DH neurons generated spontaneous EPSPs, often with spikes. Some neurons fired irregularly timed spontaneous action potentials that did not appear triggered by EPSPs. In less than 50% of the neurons the spontaneous EPSPs coincided in time with the spontaneous DR waves. The action potentials that appeared without EPSP were fired independently from DR activity. These observations confirm that elevation of interstitial free calcium concentration results in strong enhancement of excitatory transmission, especially of an EPSP of much extended duration. Virtually all neurons showed increased spontaneous activity in high [Ca2+], but only a minority appeared recruited into the synchronized discharges that are detectable as spontaneous waves in DR and VR recordings.     

Relationship between the perifornical hypothalamic area and oral pontine reticular nucleus in the rat. Possible implication of the hypocretinergic projection in the control of rapid eye movement sleep

The perifornical (PeF) area in the posterior lateral hypothalamus has been implicated in several physiological functions including the regulation of sleep–wakefulness. Some PeF neurons, which contain hypocretin, have been suggested to play an important role in sleep–wake regulation. The aim of the present study was to examine the effect of the PeF area and hypocretin on the electrophysiological activity of neurons of the oral pontine reticular nucleus (PnO), which is an important structure in the generation and maintenance of rapid eye movement sleep. PnO neurons were recorded in urethane-anesthetized rats. Extracellular recordings were performed by means of tungsten microelectrodes or barrel micropipettes. Electrical stimulation of the ipsilateral PeF area elicited orthodromic responses in both type I (49%) and type II (58%) electrophysiologically characterized PnO neurons, with a mean latency of 13.0 ± 2 and 8.3 ± 5 ms, respectively. In six cases, antidromic spikes were evoked in type I PnO neurons with a mean latency of 3.2 ± 0.4 ms, indicating the existence of PnO neurons that projected to the PeF area. Anatomical studies showed retrogradely labeled neurons in the PeF area from the PnO. Some of these neurons projecting to the PnO contained hypocretin (17.8%). Iontophoretic application of hypocretin-1 through a barrel micropipette in the PnO induced an inhibition, which was blocked by a previous iontophoretic application of bicuculline, indicating that the inhibitory action of hypocretin-1 may be due to activation of GABA_A receptors. These data suggest that the PeF area may control the generation of rapid eye movement sleep through a hypocretinergic projection by inhibiting the activity of PnO neurons.     

Intracellular study of rat substantia nigra pars reticulata neurons in an in vitro slice preparation: electrical membrane properties and response characteristics to subthalamic stimulation

The electrical membrane properties of substantia nigra pars reticulata (SNR) neurons and their postsynaptic responses to stimulation of the subthalamic nucleus (STH) were studied in an in vitro slice preparation. SNR neurons were divided into two types based on their electrical membrane properties. Type-I neurons possessed (1) spontaneous repetitive firings, (2) short-duration action potentials, (3) less prominent spike accommodations, and (4) a strong delayed rectification during membrane depolarization. Type-II neurons had (1) no spontaneous firings, (2) long-duration action potentials, (3) a prominent spike accommodation, (4) a relatively large post-active hyperpolarization, and (5) a less prominent delayed rectification. These membrane properties were very similar to those observed in substantia nigra pars compacta (SNC) neurons in slice preparations. Features common to both types of neurons include that (1) the input resistance was similar, (2) they showed an anomalous rectification during strong hyperpolarizations, and (3) they were capable of generating Ca potentials. Intracellular responses of both types of SNR neurons to STH stimulation consisted of initial short-duration monosynaptic excitatory postsynaptic potentials (EPSPs) and a short-duration inhibitory postsynaptic potential (IPSP) followed by a long-duration depolarization. The IPSP was markedly suppressed by application of bicuculline methiodide and the polarity was reversed by intracellular injection of Cl-. In the preparations obtained from internal capsule-transected rats, STH-induced EPSPs had much longer durations than those observed in the normal preparations, while the amplitude of IPSPs and succeeding small-amplitude long-duration depolarizations was small. The results indicated that SNR contains two electrophysiologically different types of neurons, and that both types of neurons receive monosynaptic EPSPs from STH and IPSPs from areas rostral to STH.     

Deafferentation Weakens Excitatory Synapses in the Developing Central Auditory System

Decreased excitatory synaptic activity during development often leads to pre- and postsynaptic atrophy, as assessed anatomically. The present study considers the effect of decreased excitatory transmission on the maturation of synaptic strength. Towards this end, cochlear nucleus neurons, which project to the ipsilateral lateral superior olive (LSO), were denervated in gerbils at postnatal day 7, before the onset of hearing. This manipulation was intended to disrupt spontaneous glutamatergic transmission in the LSO while sparing the glycinergic afferents from the medial nucleus of the trapezoid body (MNTB). Afferent-evoked synaptic activity was assessed 1–6 days after ablation in a brain slice preparation using whole-cell current- and voltage-clamp recordings. In control animals, ipsilaterally evoked excitatory postsynaptic potentials (EPSPs) were present in 91% of neurons tested, but were observed in only 60% of neurons following cochlea removal. The maximum EPSP amplitude was significantly smaller in manipulated neurons compared with controls, and this was accompanied by a higher incidence of ipsilaterally evoked inhibitory postsynaptic potentials (IPSPs). To study the efficacy of excitatory synapses in greater detail, voltage-clamp recordings were made in the presence of strychnine and AP-5 [d(O)-2-amino-5-phosphonopentanoic acid]. The minimum excitatory postsynaptic current (EPSC) amplitude, presumed to reflect the efficacy of a single glutamatergic afferent, was ～40% smaller in manipulated neurons. In contrast, MNTB-evoked IPSPs were similar in neurons from control and ablated animals. However, manipulated neurons often exhibited a rebound depolarization after a hyperpolarizing current pulse or an afferent-evoked IPSP. In 70% of manipulated neurons, synaptically evoked rebound depolarizations were reduced, but not eliminated, by glutamate receptor antagonists. The glycine receptor antagonist strychnine did eliminate the IPSP-associated depolarization in these neurons. Collectively, these results suggest that functional denervation of excitatory afferents decreases their synaptic efficacy as result of both cell loss as well as decreased strength of individual surviving synapses.