Neuroscience

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Authors: Koji Ohira & Takeshi Kaneko

Introduction

Injection of the reagents, such as virus vectors, anterograde and retrograde tracers, antagonists and agonists, into the brain is an important method in neuroscience. Since the injection site of solutions is generally restricted to a target region, such as thalamic nuclei and cortical areas, the functions, connections and morphology of the target regions can be examined by the injections of reagents.

The neocortical layer 1 contains a small number of neurons and is composed predominantly of dendritic and axonal connections (1), which receive feedback inputs from the cortex and inputs from the thalamic nuclei and other subcortical regions (2-5). Moreover, the layer 1 would exert a direct and concerted effect on the firing properties of pyramidal cells in the deep layers (6,7). Thus, the layer 1 is an important region, in which the feedforward and feedback information from higher cortical areas as well as from other lower cortical areas and subcortical regions, could be associated. Although the research on the layer 1 will become more important in the future, the injection of the regents to the layer 1 was not yet established. In this paper, we introduce the microinjection of virus vectors into the shallow neocortical layer 1.

Reagents

  1. Virus solution (8)
  2. Anesthetic agent, 7% chloral hydrate
  3. Glass capillaries (G-1, Narishige, Japan)
  4. Antibodies: primary and fluorescent-labeled secondary antibodies
  5. Tissue-Tek (Sakura Finetek, USA)
  6. Permafluor (Thermo Scientific, USA)

Equipment

  1. Dissection instruments
  2. Microloader tip (Eppendorf, Germany)
  3. Hand drill for skull penetration (Minimo, Minitor, Japan)
  4. Microgrinder (EG-44, Narishige)
  5. Puller for glass capillaries (PN-3, Narishige)
  6. Stereotaxic instrument (SR-6M, Narishige)
  7. Stereotaxic micromanipulator (SM-15, Narishige)
  8. Stereomicroscope for operation (CLS150MR, Leica, Germany)
  9. Pressure systems for injection of picoliter volumes (Picospritzer III, Intracell, UK)
  10. Cryostat (CM1850, Leica, Germany)

Procedure

Sharpening of glass needles

  1. Set glass capillaries to the puller.
  2. Pull the glass capillaries at the adequate magnet and heater strength.
  3. Mount the resulting glass needles on the needle holder.
  4. Match the grinding angle at 40°.
  5. Fill the syringe of the grinder with dH2O, which is filtered to remove dust.
  6. Sharpen the tip of the glass needle for 20 min, pouring dH2O from the syringe.
    • TIP: dH2O can protect the tip of the glass needle from the frictional heat and vibration.
  7. Observe the tip of the glass needle with microscope and make sure that the tip is sharpened and not broken.

Injection of virus solution into the neocortical layer 1

  1. Anesthetize rats or mice with chloral hydrate (350 mg/kg body weight, i.p.). It takes about 10 minutes until the animal is deeply anesthetized.
  2. Fix the head of animal to the stereotaxic instrument.
  3. Shave the surface of the head with electric hair clippers.
  4. Disinfect the dissection area with 70% ethanol.
  5. Cut the scalp at the midline sagittal suture, peel the periosteum off, and expose the skull.
  6. First put the sign on the bregma. Further, mark the insertion positions of the needle. Before marking the insertion positions, make sure precise coordinations by using a brain atlas (9).
  7. Make a hollow in the area marked on the cranial bone with a hand drill, under the observation with the stereomicroscope.
    • Caution: saline or PBS should be poured while the hollow is being made, since the frictional heat gives the brain tissue serious damage.
  8. Remove carefully the thinning skull with fine forceps. Caution: note that the blood vessels are not damaged. Especially, the hemorrhage from the vicinity of injection sites remarkably decreases the infection efficiency of virus vectors.
  9. Fill the virus solution to the glass needle, using a P2 pipetteman with a microloader tip.
  10. Attach the glass needle to the needle holder of the pressure injector.
  11. Set the needle holder to the manipulator of the stereotaxic instrument.
  12. Match the tip of the needle to the bregma: zero-correction.
  13. Set the tip of the needle to the injection point by operating the manipulator. At this time, the tip of glass needle is just set on the surface of the brain.
  14. Read the depth of the needle tip and advance the needle to the target position.
  15. Inject the solution, pushing the button of the injector. First, the injection time is set to the minimum duration, such as for 2 ms, and afterwards fine-tuned.
  16. Leave it for 10-15 min during the solution permeates the brain tissue, after the injection ends.
  17. Pull the needle out carefully.
  18. Suture the scalp after the antibiotic solution is infused to the wound.
  19. Remove the animal from the stereotaxic instrument and return it to the cage.

Immunohistochemistry

  1. Anesthetize animals with chloral hydrate (500 mg/kg body weight, i.p.).
  2. Perfuse aminals through the heart with the prewash solution (0.15 M NaCl) and then with 4% PFA in 0.1 M phosphate buffer, pH 7.4.
  3. Remove the brains and cut into blocks 5-mm thick.
  4. Immerse the tissue blocks in the same fixative at 4 oC for overnight.
  5. Transfer to 30% sucrose in PBS.
  6. Mount the tissue blocks with Tissue-Tek, frozen in a dry-ice acetone bath and store at -80 oC until use.
  7. Cut the sections at 35-50 μm with the cryostat.
  8. Wash the sections for 30 min with PBS.
  9. Preincubate with PBS containing 4% normal goat serum and 1% bovine serum albumin for 2 h.
  10. Incubate the sections for 24 h at room temperature with a primary antibody.
  11. Wash the sections for 30 min with PBS.
  12. Incubate the sections for 2 h at room temperature with a secondary fluorescent antibody.
  13. Wash the sections for 30 min with PBS.
  14. Mount the sections on glass slides and dry up.
  15. Embed the sections with Permafluor.
  16. Air-dry the specimens well.
  17. Observe the specimens with the fluorescence microscope or the confocal fluorescence microscope.

Timing

  • Sharpening of needle tips: 20 min.
  • Injection: 30 min for 1 μl of the solution.
  • Immunohisotochemistry: 4 days.

Critical Steps

Sharpening of glass needle is the most important step. Without insertion of glass needles into the neocortex without resistance, the tip of glass needle would easily pass the layer 1.

Troubleshooting

Most frequent trouble seems to be on the injection of the solution. If the solution cannot erupt, the glass needle is discarded and a new one is set. Since the diameter of tip of glass needle is quite small, the causes such as coagulation of blood make the glass needle not to erupt the solution. Thus, blood vessels should be avoided, even if they are located at the injection sites.

References

  1. Lund, J.S. & Wu, C.Q. Local circuit neurons of macaque monkey striate cortex. IV. Neurons of laminae 1-3A. J. Comp. Neurol. 384, 109-126 (1997).
  2. Glenn, L.L., Hada, J., Roy, J.P., Deschenes, M. & Steriade, M. Anterograde tracer and field potential analysis of the neocortical layer I projection from nucleus ventralis medialis of the thalamus in cat. Neuroscience 7, 1861-1877 (1982).
  3. Tigges, J. & Tigges, M. Subcortical sources of direct projections to visual cortex. In Cerebral Cortex: Visual Cortex Vol. 3 (eds. Peters, A. & Jones, E.G.) 351-378 (Plenum Press, New York, 1985).
  4. Rockland, K.S. & Virga, A. Terminal arbors of individual ‘feedback’ axons projecting from area V2 to V1 in the macaque monkey: a study using immunohistochemistry of anterogradely transported Phaseolus vulgaris leucoagglutinin. J. Comp. Neurol. 285, 54-72 (1989).
  5. Lachica, E.A. & Casagrande, V.A. Direct W-like geniculate projections to the cytochrome oxidase (CO) blobs in primate visual cortex: axon morphology. J. Comp. Neurol. 319, 141-158 (1992).
  6. Chu, Z., Galarreta, M. & Hestrin, S. Synaptic interactions of late-spiking neocortical neurons in layer 1. J. Neurosci. 23, 96-102 (2003).
  7. Shlosberg, D., Patrick, S.L., Buskila, Y. & Amitai, Y. Inhibitory effect of mouse neocortex layer I on the underlying cellular network. Eur. J. Neurosci. 18, 2751-2759 (2003).
  8. Ohira, K., et al. Ischemia-induced neurogenesis of neocortical layer 1 progenitor cells. Nat. Neurosci. In press.
  9. Paxinos, G. & Franklin, K.B.J. The mouse brain in stereotaxic coordinates (Academic Press, San Diego, 2004).

Figures

Figure 1 : Sharpening of the glass needle tip.

Fig 1

(a) An image of a glass needle before sharpening. The tip of the glass needle has obtuse angle of cross section. (b, c) The needle is difficult to be inserted into the brain tissue (b) and the tip sticks at the position, such as layer 2, where is deeper than the prediction (c). (d) An image of a sharpened needle-tip. (e, f) The sharpened needle is easily inserted into the brain tissue (e) and the needle tip is located at the desired position, layer 1 (f).

Associated Publications

Ischemia-induced neurogenesis of neocortical layer 1 progenitor cells, Koji Ohira, Takahiro Furuta, Hiroyuki Hioki, Kouichi C Nakamura, Eriko Kuramoto, Yasuyo Tanaka, Nobuo Funatsu, Keiko Shimizu, Takao Oishi, Motoharu Hayashi, Tsuyoshi Miyakawa, Takeshi Kaneko, and Shun Nakamura, Nature Neuroscience 13 (2) 173 - 179 27/12/2009 doi:10.1038/nn.2473

Author information

Koji Ohira, Division of Systems Medical Science, Institute for Comprehensive Medical Science, Fujita Health University.

Takeshi Kaneko, Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University.

Source: Protocol Exchange (2010) doi:10.1038/nprot.2010.21. Originally published online 19 January 2010.

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