Anatomy & Neurobiology

Dendritic spines are now known to be subject to use-dependent plasticity that affects both their structure and their numbers. Recently, it has been demonstrated that a use-dependent increase in the density of dendritic spines occurs in both excitatory and inhibitory ...

The aim of the research program of which the present work is a part is to understand the neural mechanisms involved in motor learning and memory. One of the mechanisms postulated to be involved in this process is the induction of long-term potentiation (LTP) in the motor cortex. LTP can be induced in motor cortical neurons by tetanic stimulation of their afferents from the somatosensory cortex. In the present study, the effects of different stimulating parameters on the induction of LTP were examined, using in-vivo, intracellular recordings from anesthetized cats. The expression of LTP was documented by measuring the amplitude and rise-time of excitatory postsynaptic potentials (EPSPs) before and after tetanic stimulation. The minimal tetanic stimulation capable of systematically inducing LTP was found to consist of a train of stimuli at 50 Hz, 5 s. Shorter trains of stimulation produced only a short-lasting, transient potentiation. In different cells, identical stimulation parameters resulted in different degrees of potentiation of synaptic responses. Following all the stimulation trains examined, EPSP amplitudes were transiently depressed before reaching potentiated levels. The duration of this depression was directly correlated with the duration and the frequency of the tetanic stimulation. In all the cells in which LTP was induced, the variability in the amplitudes of potentiated EPSP was significantly greater than that of control EPSP amplitudes. Hyperpolarization of the postsynaptic cell, during the delivery of the tetanic stimulation, inhibited the induction of LTP. These phenomena are discussed in relation to the postulated mechanisms of LTP induction in the cortex.

Disorders involving dysfunctional sensory processing are characterized by an inability to filter sensory information, particularly simultaneously arriving multimodal inputs. We examined the effects of prenatal exposure to valproic acid (VPA), a teratogen linked to sensory dysfunction, on the behavior of juvenile and adult rats, and on the anatomy of the superior colliculus, a critical multisensory integration center in the brain. VPA-exposed rats showed deficits in colliculus-dependent behaviors including startle response, prepulse inhibition, and nociceptive responses. Some deficits reversed with age. Stereological analyses revealed that colliculi of VPA-treated rats had significantly fewer parvalbumin-positive neurons, a subset of GABAergic cells.These results suggest that prenatalVPA treatment affects the development of the superior colliculus and leads to persistent anatomical changes evidenced by aberrant behavior in tasks that require sensory processing.

The laterodorsal (LD) nucleus of the thalamus has been considered a "higher order" nucleus that provides inputs to limbic cortical areas. Although its functions are largely unknown, it is often considered to be involved in spatial learning and memory. Here we provide evidence that LD is part of a hitherto unknown pathway for processing somatosensory information. Juxtacellular and extracellular recordings from LD neurons reveal that they respond to vibrissa stimulation with short latency (median = 7 ms) and large magnitude responses (median = 1.2 spikes/stimulus). Most neurons (62%) had large receptive fields, responding to six and more individual vibrissae. Electrical stimulation of the trigeminal nucleus interpolaris (SpVi) evoked short latency responses (median = 3.8 ms) in vibrissa-responsive LD neurons. Labeling produced by anterograde and retrograde neuroanatomical tracers confirmed that LD neurons receive direct inputs from SpVi. Electrophysiological and neuroanatomical analyses revealed also that LD projects upon the cingulate and retrosplenial cortex, but has only sparse projections to the barrel cortex. These findings suggest that LD is part of a novel processing stream involved in spatial orientation and learning related to somatosensory cues.

The intracortical synaptic relationships of pyramidal neurons in the cat motor cortex were studied by intracellular recording and labeling techniques. Neurons that responded with monosynaptic excitatory postsynaptic potentials (EPSPs) to microstimulation in the somatosensory cortex were identified by intracellular recordings. Long-term potentiation (LTP) was evoked in all of these neurons (n = 151, following tetanic stimulation (50 Hz, 5 s) of their afferents from the somatosensory cortex. Three of these cells (cells A-C) were identified as pyramidal neurons, following intracellular injections of Neurobiotin. The intracortical axon collaterals of these labeled cells arborized extensively, forming terminal clusters both in close proximity to the parent soma and along their long, horizontal branches. Terminal clusters in both the proximal and in the distal termination zones of each of the cells were studied by electron microscopy. In their proximal arborization zones, the axon collaterals of the labeled pyramidal neurons synapsed preferentially with dendritic spines belonging to other pyramidal cells. In contrast, in their distal terminal clusters, the axon collaterals of each of the cells formed synapses in different proportions with different postsynaptic targets. The distal axon collaterals of cell A formed 86% of their synapses with pyramidal neurons; those of cell B formed 64% of their synapses with pyramidal cells, the remaining synapses with the dendritic shafts and somata of nonpyramidal neurons, and those of cell C provided most of their output (68%) to nonpyramidal, presumably inhibitory neurons. These findings suggest a high selectivity of intrinsic axon collaterals to form specific patterns of synapses. The patterns of synaptic interactions formed by these intrinsic axon collaterals may be a substrate for shaping and modulating representation maps in the motor cortex.

Neuroanatomical tract-tracing methods were used to identify the oligosynaptic circuitry by which the whisker representation of the motor cortex (wMCx) influences the facial motoneurons that control whisking activity (wFMNs). Injections of the retrograde tracer cholera toxin subunit B into physiologically identified wFMNs in the lateral facial nucleus resulted in dense, bilateral labeling throughout the brainstem reticular formation and in the ambiguus nucleus as well as predominantly ipsilateral labeling in the paralemniscal, pedunculopontine tegmental, Kölliker-Fuse, and parabrachial nuclei. In addition, neurons in the following midbrain regions projected to the wFMNs: superior colliculus, red nucleus, periaqueductal gray, mesencephalon, pons, and several nuclei involved in oculomotor behaviors. Injections of the anterograde tracer biotinylated dextran amine into the wMCx revealed direct projections to the brainstem reticular formation as well as multiple brainstem and midbrain structures shown to project to the wFMNs. Regions in which retrograde labeling and anterograde labeling overlap most extensively include the brainstem parvocellular, gigantocellular, intermediate, and medullary (dorsal and ventral) reticular formations; ambiguus nucleus; and midbrain superior colliculus and deep mesencephalic nucleus. Other regions that contain less dense regions of combined anterograde and retrograde labeling include the following nuclei: the interstitial nucleus of medial longitudinal fasciculus, the pontine reticular formation, and the lateral periaqueductal gray. Premotoneurons that receive dense inputs from the wMCx are likely to be important mediators of cortical regulation of whisker movements and may be a key component in a central pattern generator involved in the generation of rhythmic whisking activity.

Intracellular, in vivo recordings were used to identify and subsequently to label neurons in the cat motor cortex in which long-term potentiation (LTP) was induced. Thirty-nine motor cortical neurons that produced excitatory postsynaptic potentials (EPSPs) in response to microstimulation in areas 1-2 (SI) or in area 5a (SIII) were studied. Amplitudes of EPSPs produced in response to test stimulation (1 Hz) were recorded before and after tetanic stimulation (200 Hz, 20 seconds). In 25/39 cells (64%), EPSP amplitudes were significantly increased following the tetanic stimulation (65 ? 51% average increase), and remained at the potentiated level as long as stable recordings could be maintained (20 ? 18 minutes, maximum = 90 minutes). LTP was induced exclusively in cells that produced monosynpatic EPSPs in response to area 1-2 or area 5a stimulation. Of the 39 analyzed cells, 13 were labeled by intracellular injections of 5% biocytin. Neurons in which LTP was induced included both pyramidal and nonpyramidal cells and were located exclusively in layers I1 or I11 of the motor cortex; cells in deeper cortical layers were not potentiated. These findings indicate that various corticocortical inputs can increase the efficacy of synaptic transmission in a subset of motor cortical neurons. We propose that this plasticity in synaptic transmission constitutes one of the bases of motor learning and memory.

In all sensory systems, information is processed along several parallel streams. In the vibrissato-barrel cortex system, these include the lemniscal system and the lesser-known paralemniscal system. The posterior medial nucleus (POm) is the thalamic structure associated with the latter pathway. Previous studies suggested that POm response latencies are positively correlated with stimulation frequency and negatively correlated with response duration, providing a basis for a phase locked loop-temporal decoding of stimulus frequency. We tested this hypothesis by analyzing response latencies of POm neurons, in both awake and anesthetized rats, to vibrissae deflections at frequencies between 0.3 and 11 Hz. We found no significant, systematic correlation between stimulation frequency and the latency or duration of POm responses. We obtained similar findings from recording in awake rats, in rats under different anesthetics, and in anesthetized rats in which the reticular activating system was stimulated. These findings suggest that stimulus frequency is not reliably reflected in response latency of POm neurons. We also tested the hypothesis that POm neurons respond preferentially to sensor motion, that is, they respond to whisking in air, without contacts. We recorded from awake, head-restrained rats while monitoring vibrissae movements. All POm neurons responded to passive whisker deflections, but none responded to noncontact whisking. Thus like their counterparts in the trigeminal ganglion, POm neurons may not reliably encode whisking kinematics. These observations suggest that POm neurons might not faithfully encode vibrissae inputs to provide reliable information on vibrissae movements or contacts.

Murray PD, Masri R, Keller A. Abnormal anterior pretectal nucleus activity contributes to central pain syndrome. Central pain syndrome (CPS) is a debilitating condition that affects a large number of patients with a primary lesion or dysfunction in the CNS, most commonly due to spinal cord injury, stroke, and multiple sclerosis lesions. The pathophysiological processes underlying the development and maintenance of CPS are poorly understood. We have recently shown, in an animal model of CPS, that neurons in the posterior thalamic nucleus (PO) have increased spontaneous and evoked activity. We also demonstrated that these changes are due to suppressed inhibitory inputs from the zona incerta (ZI). The anterior pretectal nucleus (APT) is a diencephalic nucleus that projects on both the PO and ZI, suggesting that it might be involved in the pathophysiology of CPS. Here we test the hypothesis that CPS is associated with abnormal APT activity by recording single units from APT in anesthetized rats with CPS resulting from spinal cord lesions. The firing rate of APT neurons was increased in spinal-lesioned animals, compared with sham-operated controls. This increase was due to a selective increase in firing of tonic neurons that project to and inhibit ZI and an increase in bursts in fast bursting and slow rhythmic neurons. We also show that, in normal animals, suppressing APT results in increased PO spontaneous activity and evoked responses in a subpopulation of PO neurons. Taken together, these findings suggest that APT regulates ZI inputs to PO and that enhanced APT activity during CPS contributes to the abnormally high activity of PO neurons in CPS.

Using the rat vibrissa system, we provide evidence for a novel mechanism for the generation of movement. Like other central pattern generators (CPGs) that underlie many movements, the rhythm generator for whisking can operate without cortical inputs or sensory feedback. However, unlike conventional mammalian CPGs, vibrissa motoneurons (vMNs) actively participate in the rhythmogenesis by converting tonic serotonergic inputs into the patterned motor output responsible for movement of the vibrissae. We find that, in vitro, a serotonin receptor agonist, ␣-Me-5HT, facilitates a persistent inward current (PIC) and evokes rhythmic firing in vMNs. Within each motoneuron, increasing the concentration of ␣-Me-5HT significantly increases the both the magnitude of the PIC and the motoneuron's firing rate. Riluzole, which selectively suppresses the Na ϩ component of PICs at low concentrations, causes a reduction in both of these phenomena. The magnitude of this reduction is directly correlated with the concentration of riluzole. The joint effects of riluzole on PIC magnitude and firing rate in vMNs suggest that the two are causally related. In vivo we find that the tonic activity of putative serotonergic premotoneurons is positively correlated with the frequency of whisking evoked by cortical stimulation. Taken together, these results support the hypothesized novel mammalian mechanism for movement generation in the vibrissa motor system where vMNs actively participate in the rhythmogenesis in response to tonic drive from serotonergic premotoneurons.

We tested the hypothesis that endogenous N-acetylaspartylglutamate (NAAG) presynaptically inhibits glutamate release at mossy fiber-CA3 synapses. For this purpose we made use of 2-(3-Mercaptopropyl)pentanedioic acid (2-MPPA), an inhibitor of glutamate carboxypeptidase II [GCP II; also known as N-acetylated alpha-linked acidic dipeptidase (NAALADase)] the enzyme that hydrolyzes NAAG into N-acetylaspartate and glutamate. Application of 2-MPPA (1 to 20 µM) had no effect on intrinsic membrane properties of CA3 pyramidal neurons recorded in vitro in whole cell current-clamp or voltage-clamp mode. Bath application of 10 µM 2-MPPA suppressed evoked EPSC amplitudes. Attenuation of EPSC amplitudes was accompanied by a significant increase in paired-pulse facilitation (50 msec inter-pulse intervals) suggesting that a presynaptic mechanism is involved. The group II metabotropic glutamate receptor (mGluR) antagonist LY341495 prevented the 2-MPPA-dependent suppression of EPSC amplitudes. 2-MPPA reduced the frequencies of TTX-insensitive miniature EPSCs (mEPSC), without affecting their amplitudes, further supporting a presynaptic action for GCP II inhibition. 2-MPPA-induced reduction of mEPSC frequencies was prevented by LY341495, reinforcing the role of presynaptic group II mGluR. Since GCP II inhibition is thought to increase NAAG levels, these results suggest that NAAG suppresses synaptic transmission at mossy fiber-CA3 synapses through presynaptic activation of group II mGluRs. Deletion of the glutamate carboxypeptidase II gene in mice reveals a second enzyme activity that hydrolyzes N-acetylaspartylglutamate. J Neurochem 83: 20-29, 2002. Bains JS, Longacher JM, and Staley KJ. Reciprocal interactions between CA3 network activity and strength of recurrent collateral synapses. Nat Neurosci 2: 720-726, 1999. Blumcke I, Behle K, Malitschek B, Kuhn R, Knopfel T, Wolf HK, and Wiestler OD. Immunohistochemical distribution of metabotropic glutamate receptor subtypes mGluR1b, mGluR2/3, mGluR4a and mGluR5 in human hippocampus. Brain Res 736: 217-226, 1996. Brager DH, Capogna M, and Thompson SM. Short-term synaptic plasticity, simulation of nerve terminal dynamics, and the effects of protein kinase C activation in rat hippocampus. J Physiol 541: 545-559, 2002. Cai Z, Lin S, and Rhodes PG. Neuroprotective effects of N-acetylaspartylglutamate in a neonatal rat model of hypoxia-ischemia. Eur J Pharmacol 437: 139-145, 2002. Canepari M and Cherubini E. Dynamics of excitatory transmitter release: analysis of synaptic responses in CA3 hippocampal neurons after repetitive stimulation of afferent fibers. Cassidy M and Neale JH. N-acetylaspartylglutamate catabolism is achieved by an enzyme on the cell surface of neurons and glia. Neuropeptides 24: 271-278, 1993. Christian EP and Dudek FE. Characteristics of local excitatory circuits studied with glutamate microapplication in the CA3 area of rat hippocampal slices. J Neurophysiol 59: 90-109, 1988. Claiborne BJ, Xiang Z, and Brown TH. Hippocampal circuitry complicates analysis of long-term potentiation in mossy fiber synapses. Hippocampus 3: 115-121, 1993. Coyle JT. The nagging question of the function of N-acetylaspartylglutamate. Neurobiol Dis 4: 231-238, 1997. Curatolo A, Marchetti M, Brancati A, and Salleo A. [Action of N-acetyl-aspartic and N-acetyl-aspartyl-glutamic acids on the spontaneous electrical activity of the cat cerebral cortex]. Arch Sci Biol (Bologna) 51: 98-103, 1967. Dobrunz LE and Stevens CF. Heterogeneity of release probability, facilitation, and depletion at central synapses. Neuron 18: 995-1008, 1997. Fuhrman S, Palkovits M, Cassidy M, and Neale JH. The regional distribution of Nacetylaspartylglutamate (NAAG) and peptidase activity against NAAG in the rat nervous system. J Neurochem 62: 275-281, 1994. Gafurov B, Urazaev AK, Grossfeld RM, and Lieberman EM. Nacetylaspartylglutamate (NAAG) is the probable mediator of axon-to-glia signaling in the crayfish medial giant nerve fiber. DA. Design, synthesis, and biological activity of a potent inhibitor of the neuropeptidase N-acetylated alpha-linked acidic dipeptidase. Jackson PF and Slusher BS. Design of NAALADase inhibitors: a novel neuroprotective strategy. Curr Med Chem 8: 949-357, 2001. Jonas P, Major G, and Sakmann B. Quantal components of unitary EPSCs at the mossy fibre synapse on CA3 pyramidal cells of rat hippocampus. J Physiol 472: 615-663, 1993. Kamiya H, Shinozaki H, and Yamamoto C. Activation of metabotropic glutamate receptor type 2/3 suppresses transmission at rat hippocampal mossy fibre synapses. J Physiol 493: 447-455, 1996. Kim J and Alger BE. Random response fluctuations lead to spurious paired-pulse facilitation. J Neurosci 21: 9608-9618, 2001. Sanabria et al. Belagaje R, Wu S, and Schoepp DD. LY341495 is a nanomolar potent and selective antagonist of group II metabotropic glutamate receptors. Neuropharmacology 37: 1-12, 1998. Kobayashi K, Manabe T, and Takahashi T. Calcium-dependent mechanisms involved in presynaptic long-term depression at the hippocampal mossy fibre-CA3 synapse. Eur J Neurosci 11: 1633-1638, 1999. Koenig ML, Rothbard PM, DeCoster MA, and Meyerhoff JL. N-acetyl-aspartylglutamate (NAAG) elicits rapid increase in intraneuronal Ca2+ in vitro. Neuroreport 5: 1063-1068, 1994. Lea PMt, Wroblewska B, Sarvey JM, and Neale JH. beta-NAAG rescues LTP from blockade by NAAG in rat dentate gyrus via the type 3 metabotropic glutamate receptor. . N-acetylaspartylglutamate protects against transient focal cerebral ischemia in rats.

The ability of rats using their whiskers to perform fine tactile discrimination rivals that of humans using their fingertips. Rats must perform these discriminations rapidly and accurately while palpating the environment with their whiskers. This suggests that whisker-derived inputs produce a robust and reliable code, capable of capturing complex, high-frequency information. The first neural representation of whisker-derived stimulus information is in primary afferent neurons of the trigeminal ganglion. Here we demonstrate that there is a continuum of directiondependent response profiles in trigeminal neurons and provide the first quantitative analysis of the encoding of complex stimuli by these neurons. We show that all classes of trigeminal ganglion neurons respond with highly reproducible temporal spike patterns to transient stimuli. Such a robust coding mechanism may allow rapid perception of complex tactile features.

REPLY: We are grateful for the comments by Ahissar et al. on our recent manuscript ) and for the opportunity to respond to these comments. Our manuscript focused on testing the hypothesis-formulated by Ahissar and collaborators-that the paralemniscal pathway in the rat's vibrissae system functions as a phase-locked loop (PLL) . We emphasize that we do not question the validity of their experimental results or of their analyses. Rather, our goal was to critically test their hypothesis that this sensory system makes use of a PLL mechanism. Two critical predictions emerge from this hypothesis: 1) response latency of neurons in the posteromedial thalamic nucleus (POm) correlates positively with whisking frequency and 2) response duration of these neurons correlates negatively with whisking frequency. In our manuscript we demonstrated that neither of these predictions holds true.