Glycinergic and GABAergic calcium responses in the developing lateral superior olive

The lateral superior olive (LSO), a binaural nucleus involved in sound localization, receives tonotopically organized inhibitory inputs from the medial nucleus of the trapezoid body (MNTB). During development, the tonotopic organization of this glycinergic/GABAergic MNTB-LSO pathway is established by activity-dependent axonal reorganization. However, the underlying mechanisms by which this reorganization takes place have remained largely unknown. As cytosolic calcium is one of the most important second messengers responsible for inducing synaptic plasticity and reorganization, we examined whether and how activity in the MNTB-LSO pathway changes the intracellular calcium concentration ([Ca2+]i) in developing LSO neurons. By applying calcium imaging techniques to Fura-2-labelled slices from neonatal rats and mice, we found that glycine and GABA (gamma-aminobutyric acid) affect [Ca2+]i in LSO neurons in an age-dependent manner; during the first postnatal week, the period at which glycine and GABA are depolarizing in the LSO, glycine and GABA always increased [Ca2+]i. However, in 2-week-old animals, the time around hearing onset when glycine and GABA are hyperpolarizing, glycine and GABA slightly decreased [Ca2+]i. Calcium responses could also be elicited by stimulation of afferent fibres from the MNTB, and these synaptic responses were mediated by glycine and GABA(A) receptors. Furthermore, GABA, which is a neurotransmitter only in the immature MNTB-LSO pathway, played a major role in generating MNTB-elicited Ca2+ responses. The direct link of glycinergic/GABAergic synaptic activity to intracellular calcium signalling during the period of inhibitory synaptic plasticity could be one of the mechanisms by which tonotopic MNTB-LSO connections become established.     

Reduction of metabotropic glutamate receptor-mediated heterosynaptic inhibition of developing MNTB-LSO inhibitory synapses

The lateral superior olivary nucleus (LSO) is an auditory relay centre within the brain stem that encodes interaural level differences for sound localization by integrating GABA/glycinergic input from the contralateral ear via the medial nucleus of the trapezoid body (MNTB), and glutamatergic input from the ipsilateral ear via the ventral cochlear nucleus (VCN). To study the development of the circuits that contribute to the establishment of sound localization, the heterosynaptic modulation mediated by glutamate released from VCN terminals and group II metabotropic glutamate receptor (mGluR) expressed on MNTB inhibitory terminals was investigated using whole-cell patch-clamp techniques. At postnatal day-4-8 (P4-8), repetitive stimulation of the VCN-LSO excitatory afferents caused significant inhibition of MNTB-LSO inhibitory postsynaptic currents (IPSCs) in amplitude with an increase of its coefficient of variation and changed the paired-pulse ratio. These effects were antagonized by LY341495, an mGluR2/3 antagonist. Thus, the suppression of MNTB-LSO synaptic responses induced by repetitive stimulation applied to the VCN-LSO glutamatergic afferent is presumably due to an activation of mGluR2/3 existing on MNTB-LSO presynaptic terminals. The suppression rate of MNTB-LSO IPSCs by DCG IV, an mGluR2/3 agonist, decreased with development and became negligible by the third week after birth. The immunohistochemical staining of mGluR2/3 in the LSO was also less apparent at P18 compared with that at P4. We suggest that mGluR-mediated heterosynaptic modulation of MNTB-LSO GABAergic/glycinergic transmission might contribute to the development of appropriate adult auditory circuits.     

An in vitro analysis of sound localization mechanisms in the gerbil lateral superior olive

One way in which animals localize sounds along the horizon is by detecting the level differences at the 2 ears. Neurons in the lateral superior olive (LSO) encode this cue by integrating the synaptic drive from ipsilateral excitatory and contralateral inhibitory connections. This synaptic integration was analyzed in 400-500-microns brain slices through the gerbil superior olive. Intracellular recordings from LSO neurons were obtained during the application of independent or conjoint electrical stimuli to the excitatory afferent and inhibitory afferent pathways. Stimulation of ascending fibers from the ipsilateral cochlear nucleus reliably evoked EPSPs and action potentials. Stimulation of the medial nucleus of the trapezoid body (MNTB) consistently evoked IPSPs. The evoked postsynaptic potentials differed in that IPSPs were 2 times the duration of EPSPs. An electrophysiological estimate of convergence indicated approximately 10 excitatory and 8 inhibitory afferents per LSO neuron. MNTB stimulation suppressed synaptically evoked action potentials. When stimulus amplitude was increased to the excitatory pathway, it was generally found that a greater MNTB stimulus was necessary to suppress the action potential. A similar commensurate rise in ipsilateral and contralateral acoustic stimulation was also found to be necessary to give the same criterion response. These results confirm that the LSO can integrate evoked action potentials and IPSPs to encode interaural level. Increasing stimulus voltage was found to decrease both action potential and IPSP latency, suggesting that intensity information may be encoded with temporal cues in the nervous system. It was also found that an evoked burst of action potentials could be inhibited in such a way as to yield intermediate discharge rates, dependent on contralateral stimulus level. Taken together, these results suggest that certain properties related to level-difference coding may be available for intracellular analysis using the brain-slice preparation. Several temporal characteristics of the synaptic potentials, including latency and duration, may play a critical role in this simple computation.     

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.     

GlyT2+ Neurons in the Lateral Cerebellar Nucleus

The deep cerebellar nuclei (DCN) are a major hub in the cerebellar circuitry but the functional classification of their neurons is incomplete. We have previously characterized three cell groups in the lateral cerebellar nucleus: large non-GABAergic neurons and two groups of smaller neurons, one of which express green fluorescence protein (GFP) in a GAD67/GFP mouse line and is therefore GABAergic. However, as a substantial number of glycinergic and glycine/GABA co-expressing neurons have been described in the DCN, this classification needed to be refined by considering glycinergic neurons. To this end we took advantage of a glycine transporter isoform 2 (GlyT2)-eGFP mouse line that allows identification of GlyT2-expressing, presumably glycinergic neurons in living cerebellar slices and compared their electrophysiological properties with previously described DCN neuron populations. We found two electrophysiologically and morphologically distinct sets of GlyT2-expressing neurons in the lateral cerebellar nucleus. One of them showed electrophysiological similarity to the previously characterized GABAergic cell group. The second GlyT2+ cell population, however, differed from all other so far described neuron types in DCN in that the cells (1) are intrinsically silent in slices and only fire action potentials upon depolarizing current injection and (2) have a projecting axon that was often seen to leave the DCN and project in the direction of the cerebellar cortex. Presence of this so far undescribed DCN neuron population in the lateral nucleus suggests a direct inhibitory pathway from the DCN to the cerebellar cortex.     

Long-lasting inhibitory synaptic depression is age- and calcium-dependent.

The developmental refinement of excitatory synapses is often influenced by neuronal activity, and underlying synaptic mechanisms have been suggested. In contrast, few studies have asked whether inhibitory synapses are reorganized during development and whether this is accompanied by use-dependent changes of inhibitory synaptic strength. The topographic inhibitory projection from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) undergoes synapse elimination during development (Sanes and Takács, 1993). To determine whether there is an associated period of synaptic plasticity, whole-cell recordings were obtained from developing LSO neurons of gerbils in a brain slice preparation. In current-clamp recordings, low-frequency stimulation of the MNTB led to a decline in IPSP amplitude by 43%. In voltage-clamp recordings, hyperpolarized LSO neurons also exhibited a long-lasting depression of MNTB-evoked inhibitory synaptic currents (34%) after low-frequency stimulation. When LSO neurons were depolarized, low-frequency stimulation of the MNTB produced a significantly larger inhibitory synaptic depression (59%). This synaptic plasticity declined dramatically by postnatal days 17-19. Similar to well studied forms of excitatory synaptic plasticity, inhibitory depression depended on postsynaptic calcium. We propose that such activity-dependent synaptic depression may support the developmental rearrangement of inhibitory terminals as they compete with neighboring excitatory and/or inhibitory inputs.     

Acoustic chiasm. III: Nature, distribution, and sources of afferents to the lateral superior olive in the cat.

The outcomes of seven experiments are reported, each directed to the nature and sources of the excitation and inhibition impinging on the lateral superior olive (LSO) in cats. In the first experiment, we used conventional 14C 2-DG methods to determine the specificity, precision, and extent of symmetry in the stimulation reaching LSO from the ipsilateral and contralateral ears. In Experiment 2, we sought the presence of GABA and glycine receptors in LSO using conventional, in vitro receptor-binding methods. On the basis of these results, we used in vitro high-affinity uptake methods in Experiment 3 to seek evidence that some of the terminals as well as the receptors in LSO are glycinergic. In Experiment 4, we used immunocytochemical methods to show that the somata known to supply the contralateral projections to LSO, and their terminals in LSO, are each immunoreactive with an antibody directed to a glycine-protein conjugate. In Experiment 5, we made use of a glycinergic neuron's avidity for transporting glycine retrogradely to label the likely sources of the glycinergic terminals in LSO. In Experiment 6, we used immunocytochemical methods to show that the spherical and globular cells of the ventral cochlear nucleus and terminals in LSO and in MTB are glutamatergic and/or aspartergic. In Experiment 7, we used receptor binding methods to determine whether the glutamate/aspartate receptors in LSO are probably of the kainate or of the quisqualate type. The results of the several experiments suggest that probably glutamate-quisqualate synapses mediate LSO's ipsilaterally driven excitatory responses and glycinergic synapses mediate its contralaterally driven inhibitory responses. The two types of input appear to be well matched in LSO's medial and middle limbs with glycinergic terminals mostly perisomatic and glutamatergic terminals mostly peridendritic. However, LSO's low frequency lateral limb appears to be somewhat different; it receives less stimulation from the contralateral ear. Instead, LSO's lateral limb may receive some of its glycinergic input directly from the ipsilateral ventral cochlear nucleus and/or indirectly via the juxtaposed lateral nucleus of the trapezoid body.     

Interleukin-1 inhibits firing of serotonergic neurons in the dorsal raphe nucleus and enhances GABAergic inhibitory post-synaptic potentials

In vitro electrophysiological data suggest that interleukin-1 may promote non-rapid eye movement sleep by inhibiting spontaneous firing of wake-active serotonergic neurons in the dorsal raphe nucleus (DRN). Interleukin-1 enhances GABA inhibitory effects. DRN neurons are under an inhibitory GABAergic control. This study aimed to test the hypothesis that interleukin-1 inhibits DRN serotonergic neurons by potentiating GABAergic inhibitory effects. In vitro intracellular recordings were performed to assess the responses of physiologically and pharmacologically identified DRN serotonergic neurons to rat recombinant interleukin-1beta. Coronal slices containing DRN were obtained from male Sprague-Dawley rats. The impact of interleukin-1 on firing rate and on evoked post-synaptic potentials was determined. Evoked post-synaptic potentials were induced by stimulation with a bipolar electrode placed on the surface of the slice ventrolateral to DRN. Addition of interleukin-1 (25 ng/mL) to the bath perfusate significantly decreased firing rates of DRN serotonergic neurons from 1.3 +/- 0.2 Hz (before administration) to 0.7 +/- 0.2 Hz. Electrical stimulation induced depolarizing evoked post-synaptic potentials in DRN serotonergic neurons. The application of glutamatergic and GABAergic antagonists unmasked two different post-synaptic potential components: a GABAergic evoked inhibitory post-synaptic potentials and a glutamatergic evoked excitatory post-synaptic potentials, respectively. Interleukin-1 increased GABAergic evoked inhibitory post-synaptic potentials amplitudes by 30.3 +/- 3.8% (n = 6) without affecting glutamatergic evoked excitatory post-synaptic potentials. These results support the hypothesis that interleukin-1 inhibitory effects on DRN serotonergic neurons are mediated by an interleukin-1-induced potentiation of evoked GABAergic inhibitory responses.     

Paired recordings from distant inhibitory neuron pairs by a sequential scanning approach

Simultaneous recordings from connected neuron pairs have brought important insights into synaptic communication between neurons. However, patch clamp recordings from neuron pairs have been largely restricted to brain areas in which connections among nearby neurons exist at a relatively high probability. In the case of more distant connections or in areas in which neurons are connected with low probability, recordings from synaptically connected neuron pairs have remained scarce. Here, we present a method that allows dual recordings from remotely connected neuron pairs by scanning potential presynaptic neurons. The applicability of this new approach was tested in the inhibitory pathway from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO), a sound localization pathway in the auditory brainstem. Using a three-step approach that sequentially combines focal uncaging of glutamate, pressure application of glutamate, and loose patch recordings allowed us to reliably achieve recordings from distant, synaptically connected GABA/glycinergic MNTB-LSO neuron pairs. Our results demonstrate that single MNTB neurons evoke highly variable mono-synaptic responses in developing LSO neurons, and heterogeneous short term synaptic dynamics, suggesting local variations in the refinement of these inhibitory connections. Paired recordings, enabled by scanning of remotely connected pairs, will be highly useful to perform detailed investigations of the synaptic function and plasticity from these circuits during the period of developmental refinement. In general, this method should provide a valuable tool to find connected neurons in other brain areas in which recording from candidate pairs has a low success rate.     

Inhibition of spinal or hypoglossal motoneurons of the newborn rat by glycine or GABA

The function of GABA or glycine during early postnatal development remains controversial as their action is reported as either excitatory or inhibitory. The present study addressed the question of the functional role of GABA or glycine on rat motoneurons shortly after birth. For this purpose, using in vitro preparations from immature rats (postnatal age, P0-P4 days), we recorded from lumbar spinal motoneurons and hypoglossal motoneurons. All data were obtained under current clamp conditions (recording with potassium methylsulphate containing electrodes) from cells at about -70 mV resting potential. On spinal motoneurons we used the glycinergic and GABAergic recurrent postsysnaptic potential (PSP) mediated by Renshaw cells to assess its impact on excitatory synaptic inputs from dorsal afferent fibres. Despite its depolarizing nature, the recurrent PSP consistently inhibited synaptic excitation of lumbar motoneurons. On hypoglossal motoneurons, exogenously applied GABA or glycine produced depolarization with decreased input resistance. This response was always associated with inhibition of cell firing induced by intracellular current pulses. Even when the membrane potential was repolarized to resting level in the presence of GABA or glycine, hypoglossal motoneurons failed to generate spikes. Conversely, similar depolarization produced by glutamate consistently facilitated spike firing. GABAergic and glycinergic synaptic potentials evoked by focal stimulation of the reticular formation inhibited firing and/or increased firing latency in the majority of hypoglossal motoneurons. These results indicate that, immediately after birth, rat motoneurons were inhibited by synaptically released or exogenously applied GABA or glycine.     

Organization of binaural excitatory and inhibitory inputs to the inferior colliculus from the superior olive

The major excitatory, binaural inputs to the central nucleus of the inferior colliculus (ICC) are from two groups of neurons with different functions-the ipsilateral medial superior olive (MSO) and the contralateral lateral superior olive (LSO). A major inhibitory, binaural input emerges from glycinergic neurons in the ipsilateral LSO. To determine whether these inputs converge on the same postsynaptic targets in the ICC, two different anterograde tracers were injected in tonotopically matched areas of the MSO and the LSO on the opposite side in the same animal. The main findings were that the boutons from MSO axons terminated primarily in the central and caudal parts of the ICC laminae but that contralateral LSO terminals were concentrated more rostrally and on the ventral margins of the MSO inputs. In contrast, the ipsilateral LSO projection converged with the MSO inputs and was denser than the contralateral LSO projection. Consistent with this finding, retrograde transport experiments showed that the very low-frequency areas of the ICC with dense MSO inputs also received inputs from greater numbers of ipsilateral LSO neurons than from contralateral LSO neurons. The results suggest that different binaural pathways through the low-frequency ICC may be formed by the segregation of excitatory inputs to ICC from the MSO and the contralateral LSO. At the same time, the ipsilateral LSO is a major inhibitory influence in the target region of the MSO. These data support the concept that each frequency-band lamina in the ICC may comprise several functional modules with different combinations of inputs.     

Tonotopic distribution and inferior colliculus projection pattern of inhibitory and excitatory cell types in the lateral superior olive of Mongolian gerbils

Sound localization critically relies on brainstem neurons that compare information from the two ears. The conventional role of the lateral superior olive (LSO) is extraction of intensity differences; however, it is increasingly clear that relative timing, especially of transients, is also an important function. Cellular diversity within the LSO that is not well understood may underlie its multiple roles. There are glycinergic inhibitory and glutamatergic excitatory principal neurons in the LSO, however, there is some disagreement regarding their relative distribution and projection pattern. Here we employ in situ hybridization to definitively identify transmitter types combined with retrograde labeling of projections to the inferior colliculus (IC) to address these questions. Excitatory LSO neurons were more numerous (76%) than inhibitory ones. A smaller proportion of inhibitory neurons were IC-projecting (45% vs. 64% for excitatory) suggesting that inhibitory LSO neurons may have more projections to other regions such the lateral lemniscus or more distributed IC projections. Inhibitory LSO neurons almost exclusively projected ipsilaterally making up a sizeable proportion (41%) of the transmitter type-labeled ipsilateral IC projection from LSO and exhibited a moderate low frequency bias (10% difference H-L). Two thirds of excitatory neurons projected contralaterally and had a slight high frequency bias (4%). One third of excitatory LSO neurons projected ipsilaterally to the IC and these cells were strongly biased toward the low frequency limb of the LSO (37%). This projection appears to be species specific in animals with good low frequency hearing suggesting that it may be a specialization for such ability.     

Vasopressin facilitates glycinergic and GABAergic synaptic transmission in developing hypoglossal motoneurons

The hypoglossal nucleus of young rats contains vasopressin binding sites and vasopressin can directly excite hypoglossal motoneurons. In addition, indirect evidence suggests that vasopressin can enhance the synaptic input to motoneurons. We have characterized this latter effect by using brainstem slices and whole-cell recordings. We found that, in the presence of blockers of fast glutamatergic transmission, vasopressin strongly facilitated inhibitory synaptic activity. On average, vasopressin caused a six-fold increase in the frequency and a 1.5-fold increase in the amplitude of GABAergic postsynaptic currents. The effect of vasopressin on glycinergic postsynaptic currents was similar in magnitude. Vasopressin did not affect the frequency of GABAergic or glycinergic miniature postsynaptic currents, indicating that the peptide-induced facilitation of inhibitory transmission was mediated by receptors located on the somatodendritic region rather than on axon terminals of presynaptic neurons. The pharmacological profile of these receptors was determined by using d[Cha4]AVP and dVDAVP, selective agonists of V1b and V2 vasopressin receptors, respectively, and Phaa-D-Tyr-(Et)-Phe-Gln-Pro-Arg-Arg-NH2, a selective antagonist of V1a vasopressin receptors. The two agonists had no effect on the frequency of inhibitory postsynaptic currents. By contrast, the antagonist suppressed the vasopressin-induced facilitation of these currents, indicating that the receptors involved were exclusively of the V1a type. Thus, vasopressin exerts a dual action on hypoglossal motoneurons: a direct excitatory action and an indirect action mediated by GABAergic and glycinergic synapses. By virtue of this dual effect, vasopressin could alter the input-output properties of these motoneurons. Alternatively, it could play a role in generating or modulating specific motor patterns.     

Modulation of dendritic synaptic processing in the lateral superior olive by hyperpolarization-activated currents

We have previously shown that mice lateral superior olive (LSO) neurons exhibit a large hyperpolarization-activated current (I(h) ), and that hyperpolarization-activated cyclic-nucleotide-gated type 1 channels are present in both the soma and dendrites of these cells. Here we show that the dendritic I(h) in LSO neurons modulates the integration of multiple synaptic inputs. We tested the LSO neuron's ability to integrate synaptic inputs by evoking excitatory post-synaptic potentials (EPSPs) in conjunction with brief depolarizing current pulses (to simulate a second excitatory input) at different time delays. We compared LSO neurons with the native I(h) present in both the soma and dendrites (control) with LSO neurons without I(h) (blocked with ZD7288) and with LSO neurons with I(h) only present peri-somatically (ZD7288+ computer-simulated I(h) using a dynamic clamp). LSO neurons without I(h) had a wider time window for firing in response to inputs with short time separations. Simulated somatic I(h) (dynamic clamp) could not reverse this effect. Blocking I(h) also increased the summation of EPSPs elicited at both proximal and distal dendritic regions, and dramatically altered the integration of EPSPs and inhibitory post-synaptic potentials. The addition of simulated peri-somatic I(h) could not abolish a ZD7288-induced increase of responsiveness to widely separated excitatory inputs. Using a compartmental LSO model, we show that dendritic I(h) can reduce EPSP integration by locally decreasing the input resistance. Our results suggest a significant role for dendritic I(h) in LSO neurons, where the activation/deactivation of I(h) can alter the LSO response to synaptic inputs.     

GABA is depolarizing in hippocampal dentate granule cells of the adolescent and adult rats

GABAergic signaling in hippocampal pyramidal neurons undergoes a switch from depolarizing to hyperpolarizing during early neuronal development. Whether such a transformation of GABAergic action occurs in dentate granule cells (DGCs), located at the first stage of the hippocampal trisynaptic circuit, is unclear. Here, we use noninvasive extracellular recording to monitor the effect of synaptically released GABA on the DGC population. We find that GABAergic responses in adolescent and adult rat DGCs are still depolarizing from rest. Using a morphologically realistic DGC model, we show that GABAergic action, depending on its precise timing and location, can have either an excitatory or inhibitory role in signal processing in the dentate gyrus.     

Trophic influences of excitatory and inhibitory synapses on neurones in the auditory brain stem.

The effects of a reduction during development of excitatory and inhibitory synaptic input on CNS neurones were studied in the lateral superior olivary nucleus (LSO) of the ferret following neonatal, unilateral cochlear removal. LSO neurones normally receive excitatory input from the ipsilateral ear and inhibitory input from the contralateral ear. After cochlear removal, the ipsilateral LSO was smaller and contained fewer neurones than either the contralateral or the normal LSO. No difference was found between the volume or number of neurones in the latter nuclei. Remaining neurones in the LSO ipsilateral to the removal were smaller than those in the contralateral LSO of the same ferrets. These data show that activity in excitatory pre-synaptic terminals can be sufficient for post-synaptic target maintenance, but that activity in inhibitory terminals alone is not.     

Glycine Receptors in Adult Guinea Pig Brain Stem Auditory Nuclei: Regulation after Unilateral Cochlear Ablation

In young adult guinea pigs, the effects of unilateral cochlear ablation were determined on the specific binding of [³H]strychnine measured in subdivisions of the cochlear nucleus (CN), the superior olivary complex, and the auditory midbrain, after 2, 7, 31, 60, and 147 postlesion days. Changes in binding relative to that in age-matched controls were interpreted as altered activity and/or expression of synaptic glycine receptors. Postlesion binding declined ipsilaterally in most of the ventral CN and in the lateral superior olive (LSO). Binding was modestly deficient in the ipsilateral dorsal CN and in the anterior part of the contralateral anteroventral CN. Binding was elevated in the contralateral LSO. Transient changes also occurred. Binding was elevated transiently, between 2 and 31 days, contralaterally in parts of the anteroventral CN, bilaterally in the medial superior olive (MSO), and bilaterally in most of the midbrain nuclei. Binding was deficient transiently, at 60 days, in most of the contralateral CN and bilaterally in the midbrain nuclei. The present findings, together with previously reported postlesion changes in glycine release, were consistent with persistently weakened glycinergic inhibitory transmission ipsilaterally in the ventral CN and the LSO and bilaterally in the dorsal CN. Glycinergic inhibitory transmission was strengthened in the contralateral LSO and transiently strengthened in the MSO bilaterally. A hypothetical model of the findings suggested that glycine receptor regulation may depend on excitatory and glycinergic input to auditory neurons. The present changes in glycine receptor activity may contribute to altered auditory functions, which often accompany hearing loss.     

GABAergic and glycinergic presympathetic neurons of rat medulla oblongata identified by retrograde transport of pseudorabies virus and in situ hybridization

Electron microscopy suggests that up to half the synaptic input to sympathetic preganglionic neurons (SPGNs) is GABAergic or glycinergic. A proportion of this input is suspected to originate from neurons located within the medulla oblongata. The present study provides definitive evidence for the existence of these supraspinal presympathetic (PS) neurons with inhibitory phenotypes. PS neurons were identified by retrograde trans-synaptic migration of pseudorabies virus (PRV) injected into the adrenal gland. GABAergic or glycinergic cell bodies were identified by the presence of glutamate decarboxylase (GAD)-67 mRNA or glycine transporter (GlyT)-2 mRNA detected with in situ hybridization (ISH). Neither GABAergic nor glycinergic PS neurons were tyrosine hydroxylase (TH)-immunoreactive (ir). GABAergic PS neurons were located within the ventral gigantocellular nucleus, gigantocellular nucleus alpha, and medial reticular formation, mostly medial to the TH-ir PS neurons. About 30% of GABAergic PS neurons were serotonergic cells located in the raphe pallidus (RPa) and parapyramidal region (PPyr). Glycinergic PS neurons had the same general distribution as the GABAergic cells, except that no glycinergic neurons were located in the RPa or PPyr and none were serotonergic. PRV immunohistochemistry combined with ISH for both GlyT2 and GAD-67 mRNAs showed that at least 63% of midline medulla GABAergic PS neurons were also glycinergic and 76% of glycinergic PS neurons were GABAergic. In conclusion, the rostral ventromedial medulla contains large numbers of GABAergic and glycinergic neurons that innervate adrenal gland SPGNs. Over half of these PS neurons may release both transmitters. The physiological role of this medullary inhibitory input remains to be explored.     

Excitatory responses in the 'direct' striatonigral pathway: effect of nigrostriatal lesion.

The existence of a striatonigral GABAergic projection that inhibits substantia nigra reticulata (SNr) cells has been well established. We report on electrophysiological evidence in the rat for a striatonigral excitatory pathway that affects 15% of all SNr cells. Using the antidromic response test to classify SNr cells in relation to their projecting nucleus, an excitatory striatonigral response was found in 57% of cells projecting to the pedunculopontine tegmental nucleus (PPTg) and 11% of cells projecting to the thalamus. SNr cells innervated by inhibitory or excitatory striatonigral inputs had a similar firing rate but a lower variation coefficient (VC) than SNr cells that did not respond to striatonigral inputs. The 6-hydroxydopamine (6-OHDA) degeneration of nigrostriatal dopaminergic cells (A9) induced an increase in the percentage of SNr cells excited from the striatum (52%) and in the VC, but no modification of the firing rate or of the number of spikes induced by each striatal stimulus. This increase in VC was found for the striatonigral inhibitory, but not for the striatonigral excitatory pathway. These data indicate that in addition to its inhibitory action, the direct striatonigral pathway has an excitatory activity that is particularly prominent for SNr cells projecting to the pedunculopontine nucleus. Because the percentage of SNr cells excited by this pathway was markedly increased by DA cell degeneration, our findings suggest that the excitatory striatonigral system could be involved in the pathophysiology of Parkinson's disease.     

Acoustic chiasm V: inhibition and excitation in the ipsilateral and contralateral projections of LSO.

When this series of experiments was begun in 1984, the activity of each lateral superior olive (LSO) in the mammalian hindbrain was known to encode the hemifield of acoustic space containing a sound source. However, the almost random bilaterality of its ascending projections seemed to jumble that identification before reaching the midbrain. At the same time, electrophysiological studies of LSO and its efferent target in the inferior colliculus, along with the strictly contralateral deficits in sound localization resulting from unilateral lesions above the level of the superior olives, indicated that hemifield allegiance was largely maintained (though reversed) at the midbrain. Here we present seven lines of biochemical evidence, some combined with prior ablations, supporting the notion that the anatomical segregation of the ipsilateral and contralateral fibers ascending from the LSO is accompanied by a corresponding segregation of their neurotransmitters: most of the ascending ipsilateral projection is probably glycinergic and, hence, inhibitory in effect, while most of the contralateral projection is probably glutamatergic/aspartergic and, hence, excitatory in effect. Taken together, the inhibitory ipsilateral projections and the excitatory contralateral projections serve to amplify functional contralaterality at the higher levels of the auditory system.